JP2004533802A - Targeted transgenic non-human mammal with human FAD presenilin mutation and reproductive offspring - Google Patents

Abstract

The present invention relates to a target transgenic non-human mammal having a gene encoding a mutant protein product of a mutated FAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation and a humanized Aβ mutation. Animals and their progeny progeny, and target transgenic non-human mammals having a mutated FAD PS-1 gene and a gene encoding a mutant protein product of the human Swedish APP695 mutation and its reproductive progeny And a method for identifying a compound useful in treating Alzheimer's disease, and a method for treating Alzheimer's disease.

Description

【０１０３】
生殖系列キメラはそれから、ＰＳ−１^{ｎＰ２６４Ｌ}を保持しているマウスの系統を樹立するために用いられた。色のついた子孫中の変異ＰＳ−１対立遺伝子の存在は、例１に記載されたものと実質的に同じ手順の後に続き、ｎｅｏ^ｒカセットを検出することを目的とするＰＣＲ戦略を使用して決定された。ＰＣＲプライマーは以下のとおりであった：ｎｅｏ２８（ＧＧＡ ＴＴＧ ＣＡＣ ＧＣＡ ＧＧＴ ＴＣＴ ＣＣ； ＳＥＱ ＩＤ ＮＯ：１３）；およびｎｅｏ４４５（ＣＣＧ ＧＣＴ ＴＣＣ ＡＴＣ ＣＧＡ ＧＴＡ ＣＧ； ＳＥＱ ＩＤ ＮＯ：１４）。ゲノムＤＮＡは尾部試料から調製された（Ｈｏｇａｎ， １９８６、前出）。４匹の色のついた子孫のうち、１匹の雌マウスがＰＳ−１^{ｎＰ２６４Ｌ}についてヘテロ接合性（ＰＳ−１^{ｎＰ２６４Ｌ／＋}）であった。すなわち、このマウスは、前述のＰＣＲ戦略に基づき、ｎｅｏ^ｒカセットについて陽性であった。同様にＰＳ−１^{ｎＰ２６４Ｌ}についてヘテロ接合性である以降の世代の子孫は、この雌を野生型の雄とかけ合わせることによって開発された。
【０１０４】
ＰＳ−１^{ｎＰ２６４Ｌ}についてヘテロ接合性（ＰＳ−１^{ｎＰ２６４Ｌ／＋}）であるマウスは、ＰＣＲに基づく方法を使用して遺伝形質を決定された。マウスＰＳ−１の野生型対立遺伝子の存在は、以下のプライマーを使って評価された：Ｘ８Ｆ（ＣＣＣ ＧＴＧ ＧＡＧ ＧＡＧ ＧＴＣ ＡＧＡ ＡＧＴ ＣＡＧ； ＳＥＱ ＩＤ ＮＯ：１５）およびＸ８Ｒ（ＴＴＡ ＣＧＧ ＧＴＴ ＧＡＧ ＣＣＡ ＴＧＡ ＡＴＧ； ＳＥＱ ＩＤ ＮＯ：１６）。これらのプライマーによる評価は１４２ｂｐのフラグメントを生じる（データは省略）。変異対立遺伝子の存在はプライマーｎｅｏ２８およびｎｅｏ４４５を使って評価され、これは４１７ｂｐのフラグメントを生じる。よって、変異についてヘテロ接合性であるマウスは、両方のバンドを生じる；変異についてホモ接合性であるマウスは４１７ｂｐのバンドのみを生じる；そして、野生型対立遺伝子についてホモ接合性であるマウスは１４２ｂｐのバンドのみを生じる。組織サンプルは動物の尾部から得られ、例１のＰＣＲ手順がそのような評価のために利用された。
【０１０５】
ＰＳ−１^{ｎＰ２６４Ｌ}対立遺伝子についてホモ接合体であるマウス（すなわちＰＳ−１^{ｎＰ２６４Ｌ／ｎＰ２６４Ｌ}）は、ヘテロ接合体のマウス（ＰＳ−１^{ｎＰ２６４Ｌ／＋}）を、ヒト化したＡＰＰ遺伝子についてホモ接合体であるマウス（ＰＣＴ公開番号Ｗ０９６／３４０９７において開示、１９９６年１０月３１日公開；これは引用することにより本明細書に完全に取り込まれている）と交雑することによって作製された。その結果生じる世代の子孫（ｇｅｎｅｒａｔｉｏｎａｌ ｏｆｆｓｐｒｉｎｇ）はそれから、ＰＳ−１^{ｎＰ２６４Ｌ}対立遺伝子についてヘテロ接合性であり、かつ、ヒト化されたＡＰＰ遺伝子についても共にヘテロ接合性であることが決定された（データは省略）；これらの世代の子孫はそれから、交雑に使用され、その結果生じる子孫がＰＳ−１^{ｎＰ２６４Ｌ}についてホモ接合性であり、同様にヒト化ＡＰＰ遺伝子についてヘテロ接合性であることが（上に概説されたＰＣＲ手順を使用して）決定された（この同腹仔からの世代の子孫は、ＰＳ−１^{ｎＰ２６４Ｌ}対立遺伝子についてヘテロ接合性／ヒト化ＡＰＰ遺伝子についてホモ接合性；ＰＳ−１^{ｎＰ２６４Ｌ}対立遺伝子についてヘテロ接合性／ヒト化ＡＰＰ遺伝子についてヘテロ接合性であるものも見出された――この同腹仔から得られた子供の数が限られていたため、二重ホモ接合体は見つからなかった）。以降の交配はＰＳ−１^{ｎＰ２６４Ｌ／ｎＰ２６４Ｌ}×ＡＰＰ^{ＮＬｈ／ＮＬｈ}マウスを生じた。
【０１０６】
ＰＳ−１^{ｎＰ２６４Ｌ}対立遺伝子についてホモ接合性のマウスはまた、ヘテロ接合性のマウス（ＰＳ−１^{ｎＰ２６４Ｌ／＋}）の交雑によっても生成された。１セットの交配で、６匹のホモ接合体が２７匹の子孫の中に見つかり、これは、ヘテロ接合体のかけ合せから予想される２５％のホモ接合体の回収率の範囲内によくおさまる。
【０１０７】
したがって、また、上に開示されたさまざまな交配アプローチに基づき、この動物の実質的に正常な生存度と胎生期生残は明らかである。
例６：ＰＧＫ−ｎｅｏカセットの切出し
ＣｒｅリコンビナーゼをコードしているｐＢＳ１８５プラスミドＤＮＡ（Ｓａｕｅｒ ｅｔ ａｌ．， Ｎｅｗ ＢｉｏＬ，１９９０， ２， ４４１−４４９、引用することにより本明細書にその全体が取り込まれている）が、前核注入（ｐｒｏｎｕｃｌｅａｒ ｉｎｊｅｃｔｉｏｎ）によって、ＰＳ−１^{ｎＰ２６１Ｌ／＋}×ＣＤ−１のかけ合わせから生成された１細胞胚内へ導入された。プラスミドは環状であったため、ゲノムへのＤＮＡの組込みは非常に低い発生頻度を有していた。Ｃｒｅリコンビナーゼを生成するＤＮＡの一過性発現は、初期胚においてＰＧＫ−ｎｅｏカセットを切り出した。注入された胚は偽妊娠の雌へと移された。ＰＧＫ−ｎｅｏカセットの切り出しは、子孫の遺伝形質を決定することによって確かめられた。これらのマウスはＰＳ−１^{Ｐ２６４Ｌ／＋}と名付けられ、ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスを生成するためにかけ合わせられた。
【０１０８】
ネオマイシン選択可能マーカーはＰＳ−１遺伝子の転写を減少させたため、Ｃｒｅリコンビナーゼの一過性発現後に起きる隣接するｌｏｘＰ部位での遺伝子組換えによって、ＰＧＫ−ｎｅｏ遺伝子が切除された。ＰＳ−１^{ｎＰ２６４Ｌ／＋}×ＣＤ−１のかけ合せによって作製された１細胞胚（ｎ ＝ １５４）は、ｐＢＳ１８５のプラスミドＤＮＡを注入され、偽妊娠の雌に移植された。ＰＧＫ−ｎｅｏ遺伝子の喪失は、例５に記述されているように、Ｘ８ＦおよびＸ８Ｒプライマーのペアを使ったＰＣＲによって、２１９塩基対フラグメントとして、子孫において評価された。ネオマイシン選択可能マーカーが切除された変異ＰＳ−１対立遺伝子は、ＰＳ−１^{Ｐ２６４Ｌ}と名付けられた。１匹の創始者マウスにおいて切り出しがうまく生じ、ヘテロ接合性（ＰＳ−１^{Ｐ２６４Ｌ／＋}）およびホモ接合性（ＰＳ−１^{Ｐ２６４Ｌ ／Ｐ２６４Ｌ}）のマウスの系統が生成された。ＰＳ−１^{Ｐ２６４Ｌ／＋}マウスは、Ｐ２６４Ｌ変異がＡβの生成および沈着に与える影響をさらに研究するために、Ｔｇ２５７６マウスおよびＡＰＰ^{ＮＬｈ／ＮＬｈ}マウスとかけ合わせられた。
例７：ノザンおよびウェスタンブロット
生後２〜６ヶ月のＰＳ−１^＋／＋、ＰＳ−１^{ｎＰ２６４Ｌ／ｎＰ２６４Ｌ}およびＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスが、ＰＳ−１のｍＲＮＡおよびタンパク質レベルを評価するために用いられた。ＲＮＡｚｏｌ Ｂ（Ｔｅｌ−Ｔｅｓｔ， Ｆｒｉｅｎｄｓｗｏｏｄ， ＴＸ）中での均質化によって、トータルＲＮＡが脳の半分から抽出された。メッセンジャーＲＮＡはＯｌｉｇｏｔｅｘカラム（Ｑｉａｇｅｎ， Ｖａｌｅｎｃｉａ， ＣＡ）によって選別された。等体積のｍＲＮＡがローディングバッファー（ＮｏｒｔｈｅｒｎＭＡＸ−Ｇｌｙ， Ａｍｂｉｏｎ， Ａｕｓｔｉｎ， ＴＸ）と混合され、５０℃に３０分間加熱され、０．７％アガロースゲル上で分離され、ナイロンメンブレンへ移された。ＰＳ−１ ｍＲＮＡは、ｐＧＥＭ−Ｔベクター（Ｐｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＷＩ）中にクローニングされたヒトＰＳ−１の３’末端（ヌクレオチド１０８３〜１４２８）をあらわす^３２Ｐ−ｄＵＴＰ標識リボプローブにより検出された。同じブロットは規準化のためにＧＡＰＤＨプローブ（Ａｍｂｉｏｎ）をハイブリダイズさせられた。ｍＲＮＡを可視化するために、メンブレンは蛍光スクリーン（ｐｈｏｓｐｈｏｒ ｓｃｒｅｅｎ）に感光され、Ｓｔｏｒｍ ８４０ Ｐｈｏｓｐｈｏｒｌｍａｇｅｒでスキャンされ、濃度測定がＩｍａｇｅＱｕａＮＴソフトウェア（Ｍｏｌｅｃｕｌａｒ Ｄｙｎａｍｉｃｓ， Ｓｕｎｎｙｖａｌｅ， ＣＡ）によって実施された。
【０１０９】
脳の半分が、１０ｍＭ Ｔｒｉｓ ｐＨ ７．４、１５０ｍＭ ＮａＣｌ、５ｍＭ ＥＤＴＡ、１％ ＳＤＳ、０．２５％デオキシコレート、０．２５％ ＮＰ−４０、およびプロテアーゼインヒビター（５ｍＭ ＰＭＳＦ、１０μｇ／ｍｌアプロチニン、１０μｇ／ｍｌロイペプチン、１０μｇ／ｍｌペプスタチン）を含む２．５ｍｌのバッファー中で均質化された（Ｌｅｅ ｅｔ ａｌ．， Ｎａｔｕｒｅ Ｍｅｄ．， １９９７， ３， ７５６−７６０）。タンパク質濃度はＢＣＡアッセイ（Ｐｉｅｒｃｅ， Ｒｏｃｋｆｏｒｄ， ＩＬ）によって決定された。全脳ライセートは還元的なローディングバッファーと混合され、３７℃で４５分間熱せられた。各サンプルの総タンパク質５０μｇが、ＮｕＰＡＧＥ １０％ポリアクリルアミドゲル（Ｎｏｖｅｘ， Ｓａｎ Ｄｉｅｇｏ， ＣＡ）上での電気泳動によって分離され、ニトロセルロースへ移された。メンブレンは０．０５％ Ｔｗｅｅｎ−２０および５％脱脂粉乳を含むＴｒｉｓ緩衝食塩水（ＴＢＳ）中、４℃で一晩ブロッキングされた。ＰＳ−１は同じ溶液で希釈されたウサギポリクローナル抗体によって検出された。Ｃ末端フラグメントは、抗体Ｂ１７．２により１：２０００の希釈で検出された（Ｄｅ Ｓｔｒｏｏｐｅｒ ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ．， １９９７， ２７２， ３５９０−３５９８）。Ｂ１７．２はヒトＰＳ−１の親水性ループドメインにあるアミノ酸残基３００〜３１５（ＥＧＤＰＥＡＱＲＲＶＳＫＮＳＫＹ； ＳＥＱ ＩＤ ＮＯ：１７）に対して作製された。Ｎ末端フラグメントは１：５００希釈のＣＰ１６０により検出された。ＣＰ１６０は、ｐＱＥ−９プラスミド（Ｑｉａｇｅｎ）を用いてバクテリア内で発現させた、６×ヒスチジンタグを付加したヒトＰＳ−１のＮ末端フラグメント（アミノ酸１〜８０）を用いて作製された。合成ＰＳ−１ Ｎ末端フラグメントは、Ｎｉ−ＮＴＡアガロース（Ｑｉａｇｅｎ）およびＳＤＳ−ＰＡＧＥを使って精製された。ペプチドはウサギへの注射のために、アクリルアミドゲルから切り出された。ＣＰ１６０のＩｇＧ画分はアフィニティー精製され、ブロッティングに使用された。一次抗体は、セイヨウワサビペルオキシダーゼ標識抗ウサギ二次抗体（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ， Ｂｅｖｅｒｌｙ， ＭＡ）により検出された。ブロットは化学発光試薬（ＬｕｍｉＧＬＯ， Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ）と反応され、Ｈｙｐｅｒｆｉｌｍ（Ａｍｅｒｓｈａｍ， Ａｒｌｉｎｇｔｏｎ Ｈｅｉｇｈｔｓ， ＩＬ）に感光された。フィルムはスキャンされ、濃度測定がＲＦＬＰ２．１ソフトウェア（Ｓｃａｎａｌｙｔｉｃｓ， Ｆａｉｒｆａｘ， ＶＡ）で実施された。
【０１１０】
ノザンブロット解析はＰＳ−１ ｍＲＮＡについて３．１ｋｂのバンドを示した。ＰＳ−１ ｍＲＮＡレベルは、ＧＡＰＤＨ ｍＲＮＡレベルに対して、ローディングの差に関して規準化された。ＰＳ−１^{ｎＰ２６４Ｌ／ｎＰ２６４Ｌ}マウス中のＰＧＫ−ｎｅｏ遺伝子の存在は、野生型レベルの２０％であるＰＳ−１ ｍＲＮＡレベルをもたらした（データは省略）。ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウス中のｍＲＮＡレベルは、ＰＳ−１^＋／＋マウスの１００％であった（データは省略）。このように、ＰＧＫ−ｎｅｏカセットの除去はＰＳ−１ ｍＲＮＡレベルを正常なレベルに戻した。
【０１１１】
ウエスタンブロッティングは、３つすべての遺伝子型において、抗体ＣＰ１６０を使用した場合には約３０ｋＤａのＮ末端ＰＳ−１フラグメントを示し、抗体Ｂ １７．２を使用した場合には約２０ｋＤａのＣ末端ＰＳ−１フラグメントを示した。抗原をあらかじめ吸収させたＣＰ１６０によるブロッティングは、約３０ｋＤａのＮ末端バンドを排除した（データは省略）。Ｃ末端ＰＳ−１フラグメントに対するＢ１７．２の特異性は、以前に示されている（Ｄｅ Ｓｔｒｏｏｐｅｒ ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ．， １９９７， ２７２， ３５９０−３５９８）。ＰＳ−１^{ｎＰ２６４Ｌ／ｎＰ２６４Ｌ}マウスにおいてはＰＳ−１ ｍＲＮＡレベルが減少していたため、ＰＳ−１タンパク質のレベルもまた正常なレベルの約１５〜２０％にまで減少していた（データは省略）。ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおける変異ＰＳ−１の正常なｍＲＮＡ発現にもかかわらず、ＰＳ−１タンパク質レベルは約５０％に減少していた（データは省略）。Ｎ末端およびＣ末端の両フラグメントは同様な程度に減少していた。これらのデータは、ＰＳ−１^{Ｐ２６４Ｌ}変異が翻訳、完全長ＰＳ−１タンパク質のプロセッシング、あるいは切断されたフラグメントの安定性に対する影響のいずれかを介して、ＰＳ−１タンパク質レベルに影響することを示している。報告は、ＰＳ−１のいくつかのＦＡＤ変異による、Ｎ末端フラグメントのさまざまな減少および／もしくはホロタンパク質の蓄積を記述した（Ｍｅｒｃｋｅｎ ｅｔ ａｌ．， ＦＥＢＳ Ｌｅｔｔ．， １９９６， ３８９， ２９７−３０３； Ｍｕｒａｙａｍａ ｅｔ ａｌ．， Ｎｅｕｒｏｓｃｉ． Ｌｅｔｔ．， １９９７， ２２９， ６１−６４； Ｌｅｖｅｙ ｅｔ ａｌ．， Ａｎｎ． Ｎｅｕｒｏｌ， １９９７， ４１， ７４２−７５３； Ｍｕｒａｙａｍａ ｅｔ ａｌ．， Ｐｒｏｇ． Ｎｅｕｒｏ−Ｐｓｙｃｈｏｐｈａｒｍａｃｏｌ． Ｂｉｏｌ． Ｐｓｙｃｈｉａｔｒ．， １９９９， ２３， ９０５−９１３； Ｔａｋａｈａｓｈｉ ｅｔ ａｌ．， Ｎｅｕｒｏｓｃｉ． Ｌｅｔｔ．， １９９９， ２６０， １２１−１２４）。対照的に、ＦＡＤ患者において、トランスフェクションを行った細胞において、およびトランスジェニックマウスもしくは遺伝子ターゲティングしたマウスにおいて、種々のＰＳ−１ ＦＡＤ変異がフラグメント形成の減少を引き起こさないことが見出された（Ｈｅｎｄｒｉｋｓ ｅｔ ａｌ．， ＮｅｕｒｏＲｅｐｏｒｔ， １９９７， ８， １７１７−１７２１； Ｌｅｅ， ｅｔ ａｌ．， Ｎａｔｕｒｅ Ｍｅｄ， １９９７， ３， ７５６−７６０； Ｐｏｄｌｉｓｎｙ ｅｔ ａｌ．， Ｎｅｕｒｏｂｉｏｌ． Ｄｉｓ．，１９９７， ３， ３２５−３３７； Ｇｕｏ ｅｔ ａｌ．， Ｎａｔｕｒｅ Ｍｅｄ，１９９９， ５， １０１−１０６； Ｌｅｖｅｓｑｕｅ ｅｔ ａｌ．， Ｍｏｌｅｃ． Ｍｅｄ， １９９９， ５， ５４２−５５４； Ｖａｎｄｅｒｈｏｅｖｅｎ ｅｔ ａｌ．， Ｎｅｕｒｏｓｃｉ． Ｌｅｔｔ．， １９９９， ２７４， １８３−１８６； Ｂｏｒｃｈｅｌｔ ｅｔ ａｌ．， Ｎｅｕｒｏｎ， １９９６，１７， １００５−１０１３； Ｄｕｆｆ ｅｔ ａｌ．， Ｎａｔｕｒｅ， １９９６， ３８３， ７１０−７１３； Ｃｉｔｒｏｎ ｅｔ ａｌ．， Ｎａｔｕｒｅ Ｍｅｄ，１９９７， ３， ６７−７２； Ｎａｋａｎｏ ｅｔ ａｌ．， Ｅｕｒ． Ｊ． Ｎｅｕｒｏｓｃｉ．， １９９９，１１， ２５７７−２５８１）。トランスフェクトしたＰＣ１２細胞における、Ｐ２６４Ｌ変異によるＮ末端フラグメント減少のレベルは、野生型ＰＳ−１の値の約３５〜４０％であり（Ｍｕｒａｙａｍａ ｅｔ ａｌ．， Ｐｒｏｇ． Ｎｅｕｒｏ−Ｐｓｙｃｈｏｐｈａｒｍａｃｏｌ． Ｂｉｏｌ． Ｐｓｙｃｈｉａｔｒ．， １９９９， ２３， ９０５−９１３）、ＰＳ−１^＋／＋マウスとの比較でＰＳ１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいて見られた減少の程度と一致した。
例８：Ａβ４０特異的および４２特異的なＥＬＩＳＡ
沈着傾向を示す（ｐｒｅｄｅｐｏｓｉｔｉｎｇ）二重遺伝子ターゲティングマウス（１〜６ヶ月のＡＰＰ^{ＮＬｈ／ＮＬｈ}マウス、５ヶ月のＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２ ６４Ｌ／＋}マウス、および１〜２ヶ月のＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウス）および、２〜４ヶ月の沈着傾向を示すＴｇ２５７６マウス（Ｈｓｉａｏ ｅｔ ａｌ．， Ｓｃｉｅｎｃｅ，１９９６， ２７４， ９９−１０２）からの脳の半分が、ドライアイス上で凍結され、−７０℃で保存された。ＰＳ−１^{Ｐ２６４Ｌ／＋}マウスとかけ合わせた、沈着傾向を示すＴｇ２５７６マウス（２〜４ヶ月のＴｇ２５７６×ＰＳ−１^＋／＋マウス、２ヵ月のＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／＋}マウス、および１ヵ月のＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウス）からの脳の半分が同様にしてさらに調製された。脳の半分は４ｍｌの０．２％ジエチルアミンおよび５０ｍＭ ＮａＣｌ中で均質化され、１００，０００×ｇで遠心分離された。上清は２ＭのＴｒｉｓ−ＨＣｌによってｐＨ ８に中和され、ＢＣＡ法（Ｐｉｅｒｃｅ， Ｒｏｃｋｆｏｒｄ， ＩＬ）によりタンパク質濃度が分析され、５％胎性クローン血清（ｆｅｔａｌ ｃｌｏｎｅ ｓｅｒｕｍ）（ＨｙＣｌｏｎｅ， Ｌｏｇａｎ， ＵＴ）と１％脱脂粉乳のＴＢＳ溶液で１：１希釈された。Ａβ４２特異的ＥＬＩＳＡは、以前に記述されたようにして実施された（Ｓａｖａｇｅ ｅｔ ａｌ．， Ｊ Ｎｅｕｒｏｓｃｉ．， １９９８， １８， １７４３−１７５２）。Ａβ４０特異的ＥＬＩＳＡは修正されて（Ｓａｖａｇｅ ｅｔ ａｌ．， Ｊ． Ｎｅｕｒｏｓｃｉ．， １９９８， １８， １７４３−１７５２）、捕捉抗体が６Ｅ１０（Ｓｅｎｅｔｅｋ， Ｎａｐａ， ＣＡ）となり、検出抗体がＡβ４０に選択的となるようにされた（ＢｉｏＳｏｕｒｃｅ Ｉｎｔｅｒｎａｔｉｏｎａｌ， Ｃａｍａｒｉｌｌｏ， ＣＡ）。ＥＬＩＳＡシグナルは、Ａβ４０もしくは４２（Ｂａｃｈｅｍ， Ｋｉｎｇ ｏｆ Ｐｒｕｓｓｉａ， ＰＡ）を用いて生成された標準曲線に基づき、総抽出タンパク質１ミリグラムあたりのＡβのナノグラム数として報告された。
【０１１２】
表２はＰＳ−１^{Ｐ２６４Ｌ}変異がＡβ沈着の出現前に、ＡＰＰ^{ＮＬｈ／ＮＬｈ}マウスの脳におけるＡβ４０およびＡβ４２のレベルに与える影響を示す。ＰＳ−１^{Ｐ２６４Ｌ}変異はＡβ４０レベルに対して有意な影響を持たなかった。１コピーのＰＳ−１^{Ｐ２６４Ｌ}変異はＡβ４２をわずかに上昇させたが、変異の影響はＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^＋／＋マウスと比較して、ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいてのみ有意であった。Ａβ４２レベルにおけるこの増加は、ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^＋／＋およびＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／＋}マウスと比較して、ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおけるＡβ４０に対するＡβ４２の比に有意な上昇を引き起こした。Ｔｇ２５７６マウスはＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスよりも著しく多いＡβ４０およびＡβ４２を有していたが、Ａβ４２／４０比はＴｇ２５７６とＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^＋／＋マウスにおいて同様であった。
【０１１３】
表３は、ＰＳ−１^{Ｐ２６４Ｌ}変異がＡβ沈着の出現前に、Ｔｇ２５７６マウスの脳におけるＡβ４０およびＡβ４２レベルに対して与える影響を示している。ＰＳ−１^{Ｐ２６４Ｌ}変異はＡβ４０レベルに対して有意な影響を持たなかった。１コピーのＰＳ−１^{Ｐ２６４Ｌ}変異はＡβ４２をわずかに上昇させたが、変異の影響はＴｇ２５７６×ＰＳ−１^＋／＋マウスと比較して、Ｔｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいてのみ有意であった。このＡβ４２レベルの増加は、Ｔｇ２５７６×ＰＳ−１^＋／＋マウスと比較してＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいて、Ａβ４０に対するＡβ４２の比率に有意な上昇を引き起こした。このように、ＰＳ−１^{Ｐ２６４Ｌ}変異がＡβレベルに与える影響はＴｇ２５７６とＡＰＰ^{ＮＬｈ／ＮＬｈ}マウスについて同様であった。
【０１１４】
【表２】

【０１１５】
【表３】

【０１１６】
例９：免疫組織化学および組織学
ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスが生後１２ヵ月目に調べられた。生後３、６、９、１２、１５および１８ヵ月のＰＳ−１^＋／＋、ＰＳ−１^{Ｐ２６４Ｌ／＋}もしくはＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}であるＡＰＰ^{ＮＬｈ／ＮＬｈ}マウスが評価された。調べられたさらなるマウスはＴｇ２５７６であり、ＰＳ−１^＋／＋、ＰＳ−１^{Ｐ２６４Ｌ／＋}もしくはＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}で、生後１、２、４、６、９、１２、１５および１８ヵ月であった。Ｃ５７Ｂ６／ＳＪＬマウスへのかけ合わせによって維持された他のＴｇ２５７６マウスもまた、生後６、９、１２、１５、１８および２１ヶ月目に調べられた。マウスはリンガー溶液を灌流され、脳が取り除かれ、二等分された。各脳の半分は７０％エタノールと１５０ｍＭ ＮａＣｌに４８時間浸され、パラフィンが包埋され、矢状面で１０μｍに切片化された。１６切片のセットが２００μｍの間隔で取られ、免疫組織化学によってＡβの沈着を示すために染色された。使用された抗体は、ヒトＡβのアミノ酸１〜２８に対して作製されたウサギポリクローナル抗体の１１５３（Ｓａｖａｇｅ ｅｔ ａｌ．， Ｎｅｕｒｏｓｃｉｅｎｃｅ， １９９４， ６０， ６０７６１９）とモノクローナル抗体４Ｇ８および６Ｅ１０（Ｓｅｎｅｔｅｋ）であった。切片は４Ｇ８の場合は８０％の蟻酸で前処理され、１１５３および６Ｅ１０の場合は前処理を行わず、そして、１：１，０００希釈で一次抗体と共に一晩反応させられた。抗体はビオチン化された二次抗体（１：１００）を用いて複合体化され、セイヨウワサビペルオキシダーゼ標識ストレプトアビジン（ＢｉｏＧｅｎｅｘ， Ｓａｎ Ｒａｍｏｎ， ＣＡ）を用いて連結され、ニッケル増強３，３’−ジアミノベンンジデンを用いて可視化された。非トランスジェニックマウス、およびあらかじめ吸収された一次抗血清が染色の対照として役立った。切片のさらなるセットは、チオフラビンＳを用いて染色され、蛍光顕微鏡で調べられた。
【０１１７】
ＣａｓｔＧｒｉｄシステム（Ｏｌｙｍｐｕｓ， Ｃｏｐｅｎｈａｇｅｎ， Ｄｅｍｎａｒｋ）を使用し、１１５３抗体で染色された１セットの１６切片において、新皮質におけるプラーク量（ｐｌａｑｕｅ ｌｏａｄ）が定量された。新皮質の体積とＡβ沈着によって占められる新皮質のパーセント体積は、点計数（ｐｏｉｎｔ ｃｏｕｎｔｉｎｇ）（Ｗｅｉｂｅｌ ｅｔ ａｌ．， （１９７９） Ｓｔｅｒｅｏｌｏｇｉｃａｌ ｍｅｔｈｏｄｓ， ｖｏｌ． １： ｐｒａｃｔｉｃａｌ ｍｅｔｈｏｄｓ ｆｏｒ ｂｉｏｌｏｇｉｃａｌ ｍｏｒｐｈｏｍｅｔｒｙ， ４１５ ｐｐ． Ｌｏｎｄｏｎ： Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ）によって、立体解析学的に決定された。代表的な結果が表４に示されている。
【０１１８】
表４．生後６ヶ月の新皮質におけるＡβプラーク量（％体積含有率）

細胞外のＡβ沈着は、ＰＳ−１^＋／＋のものと比較して、ＰＳ−１^{Ｐ２６４Ｌ／＋}もしくはＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}のＴｇ２５７６マウスにおいては著しく加速されていた。Ｔｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／＋}マウスにおいては、Ａβ沈着は生後２ヵ月では認められなかったが、生後４ヵ月では存在していた。Ｔｇ２５７６ ｘ ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスでは、Ａβ沈着は生後１ヵ月では存在しなかったが、生後２ヵ月では存在していた。Ｔｇ２５７６×ＰＳ−１^＋／＋マウスは生後６ヵ月までＡβ沈着を示さず、その量は、Ｃ５７Ｂ６／ＳＪＬバックグラウンドで維持された生後６ヵ月のＴｇ２５７６マウスにおいて見られるそれに相当した。１１５３抗体で可視化されたＡβプラーク量は、より高齢のＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／＋}およびＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスの新皮質において、劇的に増加していた（データは省略）。生後４ヶ月のＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／＋}マウスおよび生後２ヶ月のＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスは、４Ｇ８もしくはチオフラビンＳで染色される多数の沈着を有しており、これは最早期の沈着が緻密な原線維アミロイドを含むことを示している。
【０１１９】
Ｔｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいて見られる沈着の加速に加えて、もう一つの差異は沈着の領域分布であった。生後６ヵ月のＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウス、生後９ヵ月のＴｇ２５７６×ｐＳ−１^{Ｐ２６１Ｌ／＋}マウスおよび生後１８ヵ月のＴｇ２５７６（Ｃ５７Ｂ６／ＳＪＬバックグラウンド）マウスを比較すると、沈着の密度は、終脳構造において類似していた（データは省略）。しかし、Ｔｇ２５７６×ｐＳ−１^{Ｐ２６１Ｌ／ＰＩ６１Ｌ}マウスの皮質下構造では、Ａβ沈着の量がＴｇ２５７６×ＰＳ−１^{Ｐ２６４Ｌ／＋}およびＴｇ２５７６マウスにおけるよりも、非常に多くなっていた（データは省略）。
【０１２０】
ＡＰＰ^{ＮＬｈ／ＮＬｈ}マウスにおけるＡβ沈着は、生後２２ヵ月まで評価された。沈着の証拠は発見されなかった。同様に、マウスＡＰＰについて野生型であるＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいて、生後１２ヶ月で、沈着は見つからなかった。極めて稀なＡβ沈着が、１１５３抗体およびチオフラビンＳの両方を用いて、ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／＋}マウスの皮質において生後１２ヶ月に認められた。生後１８ヵ月では、これらのマウスにおけるＡβ沈着は、より多数で、より大きかった。
【０１２１】
２コピーのＰＳ−１^{Ｐ２６４Ｌ}変異は、ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおけるＡβ４２の増加をもたらし（表２）、早い年齢でのＡβ沈着をもたらした。ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスでは、Ａβ沈着は、生後３ヵ月では見つからなかったが、６ヵ月では存在していた。沈着は、ＡＰＰ^{ＮＬｈ／ＮＬｈ}×ＰＳ−１^{Ｐ２６４Ｌ／Ｐ２６４Ｌ}マウスにおいて、加齢とともに増加した。
【０１２２】
本文書において引用もしくは記述された各特許、特許出願、および出版物の開示は、この結果、引用することにより、それらの全体において、本明細書に取り込まれている。
【０１２３】
ここに記述されたものに加えて、前の記述から本発明のさまざまな修正が当業者にとって明らとなるであろう。そのような修正はまた、添付特許請求の範囲の範囲内におさまることが意図されている。
【図面の簡単な説明】
【図１】
発明にかかる、遺伝子ターゲッティングの一般的原理を具体的に説明する図解である。
【図２】
発明にかかる、フラッシュ［Ｆｌａｓｈ］（商標）非放射活性遺伝子マッピングキットを使用して調製された、一組のマウスＰＳ−１ゲノムクローン地図である。制限エンドヌクレアーゼの文字略語は以下のとおりである：Ｅ、ＥｃｏＲＩ；Ｘ、ＸｂａＩ；Ｈ、ＨｉｎｄＩＩＩ；Ｂ、ＢａｍＨＩ；Ｘｈ、ＸｈｏＩ。
【図３】
発明にかかる、フラッシュ［Ｆｌａｓｈ］（商標）制限マッピング法を具体的に説明するのに使用される代表的制限地図である。
【図４】
発明にかかる、ＰＳ−１の制限地図上にエキソン７および８を位置づけるための戦略を具体的に説明する図である。
【図５】
発明にかかる、ＰＳ−１のエキソン８とｐＰＳ１−８−ＴＶ置換ベクターとの間の関係を具体的に説明する一対の遺伝子地図である。制限エンドヌクレアーゼの文字略語は以下のとおりである：Ｅ、ＥｃｏＲＩ；Ｘ、ＸｂａＩ；Ｈ、ＨｉｎｄＩＩＩ；Ｂ、ＢａｍＨＩ；Ｘｈ、ＸｈｏＩ、Ｎ、ＮｏｔＩ。
【図６】
発明にかかる、プラスミドｐＰＮＴＩｏｘ^２の構築を具体的に説明する図解である。
【図７】
発明にかかる、プラスミドｐＰＳ１−ＸＨ１６の構築を具体的に説明する図解である。
【図８】
発明にかかる、プラスミドｐＰＳ１−ＸＢ１の構築を具体的に説明する図解である。
【図９】
発明にかかる、プラスミドｐＰＳ１−Ｘ１５およびｐＰＳ１−Ｘ２の構築を具体的に説明する図解である。
【図１０】
発明にかかる、プラスミドｐＰＳ１−Ｘ３１９の構築を具体的に説明する図解である。
【図１１】
発明にかかる、プラスミドｐＰＳ１−Ｘ１５およびｐＰＳ１−Ｘ２からの相同な５’アームの制限マッピングを具体的に説明する図解である。
【図１２】
発明にかかる、ＰＳ１の相同な３’および５’アームについての一対の制限地図である。
【図１３】
発明にかかる、本発明のＰ２６４Ｌ突然変異およびＡｆｌＩＩ制限エンドヌクレアーゼ部位の付加を遂げるための塩基変化を具体的に説明するＰＳ−１のエキソン８の部分的配列である。
【図１４】
発明にかかる、プラスミドｐＰＳ１−ＸＢ８５の構築を具体的に説明する図解である。
【図１５】
発明にかかる、プラスミドｐＰＳ１−２０６の構築を具体的に説明する図解である。
【図１６】
発明にかかる、プラスミドｐＰＳ１−３６０の構築を具体的に説明する図解である。
【図１７】
発明にかかる、プラスミドｐＰＮＴ３’４１３の構築を具体的に説明する図解である。
【図１８】
発明にかかる、プラスミドｐＰＳ１−８−ＴＶの構築を具体的に説明する図解である。
【図１９】
発明にかかる、マウスＰＳ１内の相同的組換えを検出するための戦略を具体的に説明する図解である。制限エンドヌクレアーゼの文字略語は以下のとおりである：Ｅ、ＥｃｏＲＩ；Ｘ、ＸｂａＩ；Ｎ、ＮｏｔＩ；Ｈ、ＨｉｎｄＩＩＩ；Ｂ、ＢａｍＨＩ；Ａ、ＡｐａＩ；Ａｆ、ＡｆｌＩＩ；Ｓｃ、ＳｃａＩ；Ｋ、ＫｐｎＩ；およびＨｃ、ＨｉｎｃＩＩ。[0001]
(Relationship with related applications)
This application is a continuation-in-part application and is described in U.S. Pat. No. 6,057,069, filed Aug. 29, 1997. S. C. See U.S. Patent Application Serial No. 09 / 041,185, filed March 10, 1998, claiming priority under section 35 § 119 (e). S. C. Claims of priority under Article 35 §120, each of which is incorporated herein by reference in its entirety.
[0002]
(Field of the Invention)
The present invention relates to a gene-targeted non-human mammal comprising a human mutation in the presenilin 1 (PS-1) FAD gene of a non-human mammal, a compound for treating Alzheimer's disease. The present invention relates to an identification method and a method for treating Alzheimer's disease.
[0003]
(Background of the Invention)
Alzheimer's disease (AD) is an age-dependent neurodegenerative disorder that leads to significant behavioral changes and dementia. Prominent pathologies include atrophy of brain gray matter as a result of considerable loss of neurons and synapses, and protein deposition in the form of both intraneuronal neurofibrillary tangles and extracellular amyloid plaques in the brain parenchyma I do. In addition, affected areas of the brain in AD exhibit reactive gliosis, which appears to be a response to brain injury. The surviving neurons from the population susceptible to AD include the cytoskeleton (Wang et al., Nature Med., 1996, 2, 871-875), the Golgi complex (Salehi et al., J. Neuropath. Exp. Neurol., 1995, 54, 704-709) and alterations in the endosomal-lysosomal system (Cataldo et al., Neuron, 1995, 14, 671-680) show signs that the metabolism is at risk.
[0004]
Approximately 10 to 30% of AD cases are inherited in an autosomal dominant manner and are termed "familial Alzheimer's disease" or "FAD." Genetic linkage studies have shown that FAD is heterogeneous and the majority of cases are associated with genetic mutations on chromosomes 1, 14, 19 or 21 (Siman and Scott, Curr. Opin. Biotech., 1996, 7, 601-607). Importantly, these individuals develop the classic symptomatic and pathological features of the disease, confirming that the mutation is associated with the development of the disease rather than the associated syndrome It has been shown. The locus on chromosome 14 is associated with a significant portion of FAD, and the mutation at that locus is named "S182" or "Presenilin 1" (PS-1), which encodes a 467 amino acid protein. (Sharrington et al., Nature, 1995, 375, 754-760; Clark et al., Nature Genet., 1995, 11, 219-222). The closely related gene "STM2" or "Presenilin 2" (PS-2), located on chromosome 1, is a member of two additional FAD families that include German family offspring from the Volga basin in Russia. (Levy-Lahad et al., Science, 1995, 269, 973-977; Rogaev et al., Nature, 1995, 376, 775-778). PS-1 and PS-2 share a total of 67% amino acid sequence homology, and primary structural analysis indicates that they are integral membrane proteins with 6 to 8 transmembrane domains ( Slunt et al., Amyloid-Int. J. Exp. Clin. Invest., 1995, 2, 188-190; Doan et al., Neuron, 1996, 17, 1023-1030). Much of the information regarding the function of presenilin is termed "SEL-12" (a 6-8 transmembrane protein that appears to participate in the intracellular signaling pathway mediated by the lin-12 / glp-1 / Notch family). C. Arising from the identification of presenilin homologs in C. elegans (Levitan and Greenwald, Nature, 1995, 377, 351-354). The PS-1 and SEL-12 proteins share 49% sequence homology and have similar membrane orientation. Importantly, both human PS-1 and PS-2 are C. Mutants in C. elegans can rescue the sel-12 phenotype, indicating a role for presenilin in Notch signaling (Levitan et al., Proc. Natl. Acad. Sci. USA, 1996). , 93, 14940-14944).
[0005]
FAD associated with presenilin is highly penetrating, and the aggressive nature of the disease suggests that the mutant protein is involved in the pathogenesis of AD pathology. To date, more than 70 FAD mutations have been identified in PS-1, and three FAD mutations have been found in PS-2. Most of the FAD mutations are in conserved positions between the two presenilin proteins, suggesting that they affect functionally or structurally important amino acid residues. Interestingly, many of the mutated amino acids are also conserved in SEL-12. All but two of the presenilin mutations are missense mutations. One exception results in aberrant RNA splicing events that eliminate exon 9 and create an internally deleted mutant protein (Perez-Tur et al., NeuroReport, 1995, 7, 297-301; Sato et al. Hum. Mutat. Suppl., 1998, 1, S91-94; and Prihar et al., Nature Med., 1999, 5, 1090). Others result in two deletion transcripts (Δ4 and Δ4cryptic) and one full-length transcript with the amino acid Thr inserted between 113 and 114 (DeJonghe et al., Hum. Molec. Genet). , 1999, 8, 1529-1540). The latter transcript leads to the pathophysiology of AD. These latter points indicate that, together with the genetic dominance of the disease, the etiology of the disease in the presenilin family has novel and detrimental functions rather than simple loss or reduction of normal presenilin levels. Requires the production of a presenilin protein. The mutation, however, appears to disrupt normal presenilin function because the mutant presenilin is not able to rescue or completely rescue the sel-12 phenotype (Levitan et al., Proc. Natl. Acad. USA, 1996, 93, 14940-14944).
[0006]
Presenilin expression profiles have been examined at an approximate level, but to date, however, these analyzes have yielded little information about the mechanisms of disease pathogenesis. Both presenilin 1 and 2 are widely expressed in the CNS and peripheral tissues. In the brain, expression is enhanced by neurons, but is evident in both areas susceptible and resistant to AD. Cell localization studies indicate that the protein accumulates primarily in the Golgi complex and the endoplasmic reticulum, but no significant changes in expression levels or subcellular distribution have been attributed to FAD mutations. (Kovacs et al., Nature Med., 1996, 2, 224-229).
[0007]
Presenilin proteins are proteolytically processed by two intracellular pathways. Under normal conditions, accumulation of a 30 kD N-terminal and a 20 kD C-terminal proteolytic fragment occurs in the absence of the full length protein. This processing pathway appears to be highly regulated and relatively slow, explaining the turnover of only a small portion of the full-length protein. The remaining portion appears to be rapidly degraded by the proteasome in the alternative pathway (Thinkaran et al., Neuron, 1996, 17, 181-190; Kim et al., J. Biol. Chem., 1997, 272, 1106-111010). ). The proteolytic metabolism of PS-1 variants associated with FAD appears to be different, but the relevance of the change to etiology is unknown (Lee et al., Nature Med., 1997, 3, 756). -760).
[0008]
One pathological role of the mutant presenilin in FAD is its effect on the processing of amyloid precursor protein (APP) and the production of Aβ peptide, a major protein component of extracellular neuritic plaques in the brain of AD. It seems to be related to. Elevated serum concentrations of the longer form of Aβ (Aβ42), which is considered a more pathogenic species of Aβ peptide, have been measured in patients carrying the PS-1 and PS-2 mutations (Scheuner et al.). , Nature Med., 1996, 2, 864-870). In addition, FAD brains with PS-1 mutations have abundant Aβ deposits (Lemere et al., Nature Med., 1996, 2, 1146-1150; Mann et al., Ann. Neurol., 1996, 40, 149). Gomez-Isla et al., Ann. Neurol., 1997, 41, 809-813). Elevated levels of Aβ 1-42 were also found in cells transfected with mutant PS-1 or PS-2, and in mice expressing mutant PS-1 (Borchelt et al. Neuron, 1996, 17, 1005-1003; Duff et al., Nature, 1996, 383, 710-713; Citron et al., Nature Med., 1997, 3, 67-72; Murayayama et al., Prog. Neuro-Psychopharmacol. Biol. Biol. Murayama et al., Neurosci. Lett., 1999, 265, 61-63; Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581). The mechanism by which the mutant presenilin affects the processing of APP is not known, but these results support a role responsible for increased Aβ42 production in the development of FAD. Importantly, the processes by which mutant presenilin contribute to other AD pathologies, such as putative intracellular signaling and cell death pathways that are directly or indirectly involved in neuronal dysfunction and degeneration Can be affected as well.
[0009]
Genetically engineered animals have been used extensively to examine the function of certain gene products in vivo and their role in the development of disease phenotypes. Mouse genetic engineering involves at least two distinct approaches: (1) a transgenic approach in which exogenous genes are randomly inserted into the host genome; and (2) specific endogenous DNA sequences or genes. Can be achieved according to a gene targeting approach in which is partially or completely removed or replaced by homologous recombination. The transgene of a transgenic organism is generally composed of a DNA sequence encoding the protein sequence and a promoter that directs the expression of the protein coding sequence. Transgenic organisms express the transgene in addition to all normally expressed native genes. On the other hand, the target gene of the target transgenic animal can be obtained in two ways: (1) a functional form in which the modified version of the target gene is expressed, or (2) a target gene that is disrupted. It can be modified in one of the non-functional or "null" forms, resulting in loss or reduction of expression. If the target gene is a single copy gene and the animal is homozygous at the target locus, depending on the type of modification, the animal will either not express the target gene or will Either express a modified version of the target gene only in the presence.
[0010]
Transgenic mice expressing native and mutant forms of presenilin protein have been described (Borchelt et al., Neuron, 1996, 17, 1005-1003; Duff et al., Nature, 1996, 383, 710-713; Borchelt). Et al., Neuron, 1997, 19, 939-945; Citron et al., Nature Med., 1997, 3, 67-72; Chui et al., Nature Med., 1999, 5, 560-564; and Nakano et al., Eur. Neurosci., 1999, 11, 2577-2581). Although mice carrying the mutation in PS-1 had elevated levels of Aβ 1-42, they did not form Aβ deposits characteristic of AD or were associated with AD Shows no behavioral defects. Neuronal loss has been described by one group (Chui et al., Nature Med., 1999, 5, 560-564). When transgenic mice carrying the PS-1 mutation were crossed with transgenic mice carrying the Swedish APP mutation, there was a marked enhancement of the formation of Aβ deposits (Borchelt et al., Neuron, 1997, 19). Holcomb et al., Nature Med., 1998, 4, 97-100; Lamb et al., Nature Neurosci., 1999, 2, 695- 697). Targeted recombinant PS-1 null mice lacking one or both functional alleles of the PS-1 gene have also been described (Wong et al., Nature, 1997, 387, 288-292 and Shen et al., Cell, 1997; 89, 629-639). Mice in which both PS-1 alleles have been disrupted resulting in complete loss of PS-1 expression are not viable and die shortly after birth. Abnormal phenotype or altered processing of APP has not been reported in mice lacking only one of the two PS-1 alleles, but inhibition of APP processing is found in neurons from PS-1 null mice (DeStrooper et al., Nature, 1998, 391, 387-390).
[0011]
In this application, a gene targeting approach to generating AD models (Reaume et al., J. Biol. Chem., 1996, 271, 23380-23388, which is hereby incorporated by reference in its entirety) is described. . One model uses the Swedish FAD mutation in the APP gene and the "humanized" mouse Aβ sequence (US Pat. No. 5,777,194, which is hereby incorporated by reference in its entirety). )). This mouse (APP^{NLh / NLh}) Produced normal levels of APP and overproduced human Aβ1-40 and Aβ1-42, but did not deposit Aβ (Reaume et al., J. Biol. Chem., 1996, 271, 23380-23388). ). A human PS-1 mutation, particularly the P264L mutation, was introduced into the mouse PS-1 gene. The P264L mutation is a non-conservative amino acid substitution in a cluster of mutations in exon 8, causing the onset of FAD in the mid-40s to mid-50s (Campion et al., Hum. Molec. Genet., 1995). Wasco et al., Nature Med., 1995, 1, 848). Mating is APP^{NLh / NLh}× PS-1^{P264L / P264L}Dual target transgenic mice were generated. These mice had elevated levels of Aβ 1-42 sufficient to cause Aβ deposition. PS-1^P264LMice carrying the mutation were also crossed with Tg2576 mice that overexpress the Swedish APP695 (Hsiao et al., Science, 1996, 274, 99-102; available from Mayo Clinic, Rochester, MN). One distinct advantage of the present invention is that the stringency of the protein expression pattern is maintained for heterozygous and homozygous target transgenic mice. Because the expression is under an endogenous promoter. Furthermore, the expression level of the whole protein (holoprotein) cannot be changed.
[0012]
(Summary of the Invention)
The present invention provides a target recombinant comprising a gene encoding a mutated FAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation, and a mutant protein product of a humanized Aβ mutation. It relates to non-human mammals, as well as their generational progeny. The present invention also provides a target transgenic non-human mammal comprising a mutated FAD presenilin-1 (PS-1) gene and a gene encoding a mutant protein product of the human Swedish APP695 mutation, and It also relates to its reproductive offspring. Preferably, the PS-1 gene has been mutated to contain a human P264L mutation (Wasco et al., Nature Medicine, 1995, 1,848). In particular, the present invention relates to mice wherein a portion of the mouse presenilin 1 gene encoding the presenilin 1 protein has been replaced with a DNA sequence resulting in a mouse presenilin 1 gene containing a human mutation, most preferably a P264L mutation. More specifically, the nucleotide sequence of codon 264 of the mouse presenilin 1 gene is changed from CCG to CTT (which is a nucleotide sequence found to constitute a human P264L mutation). The mutated gene codon encodes leucine instead of proline at amino acid number 264 of presenilin 1. In addition, and even more specifically, one nucleotide base in codon 265 of the mouse presenilin 1 gene is changed from adenosine to guanosine, but this change does not result in an amino acid change in the expressed protein. However, the combined sequence of codons 264 and 265 creates a restriction enzyme site for the restriction enzyme AflII after incorporation of the most favorable changes described above.
[0013]
Thus, in one embodiment, the present invention relates to a mouse presenilin 1 protein comprising a human mutation, most preferably a P264L mutation, in place of the mutated FAD gene, preferably the sequence encoding the native presenilin 1 protein. It features non-human mammals and reproductive offspring homozygous for the target mutant PS-1 gene comprising the coding sequence. In another aspect, the invention provides a mouse presenilin 1 protein that contains a human mutation, most preferably a P264L mutation, in place of the mutated mouse FAD gene, preferably a sequence encoding the native presenilin 1 protein. Non-human mammals and reproductive offspring heterozygous for the target PS-1 gene comprising the following sequence:
[0014]
The invention also relates to a mammal heterozygous or homozygous for the mutation of the PS-1 gene and the human Swedish APP695 or progeny progeny thereof, or a mutation of the PS-1 gene, human FAD Swedish Administering the compound to a mammal heterozygous or homozygous for the mutation and the humanized Aβ mutation and its reproductive offspring, and determining the amount of Aβ42 peptide in a tissue sample from the mammal. A method of identifying a compound for treating Alzheimer's disease, comprising:
[0015]
The present invention is also directed to a method of treating an Alzheimer's disease comprising administering to an individual suspected of having Alzheimer's disease an effective Alzheimer's disease therapeutic amount of a compound identified by the above-described method. Can be
[0016]
The present invention is also directed to compounds identified by any of the methods described above.
[0017]
(Detailed description)
The present invention relates to a target transgenic non-human mammal comprising a presenilin 1 gene comprising a human mutation, most preferably a human P264L mutation, in the endogenous (ie, native) genome of the non-human mammal. (And reproductive offspring of such mammals). Non-human mammals can also include a human FAD Swedish mutation and a humanized Aβ mutation. The non-human mammal can also contain a human Swedish APP695 mutation in addition to the human PS-1 mutation. Most preferably, the target transgenic non-human mammal produces a mutated presenilin 1 protein in place of the presenilin 1 protein normally produced by the non-human mammal. Target transgenic non-human mammals homozygous for the presenilin 1 gene containing a human mutation, such as the human P264L mutation, exclusively produce a mutated presenilin 1 protein. Target transgenic non-human mammals heterozygous for the presenilin 1 gene containing a human mutation, such as the human P264L mutation, produce both the mutated presenilin 1 protein and the native presenilin 1 protein. Preferably, the target transgenic non-human mammal of the invention is a rodent, and more specifically, a mouse.
[0018]
Importantly, since the non-human mammals of the present invention are produced by gene targeting as opposed to transgenic technology, the mammals will be able to replace the mutated presenilin 1 protein with the normal endogenous presenilin 1 protein. Produced exclusively by the expression mechanism of Advantageously, and also, unlike expression from a transgenic approach, the presenilin 1 protein is expressed from a gene with a normal copy number and under the control of the endogenous presenilin 1 gene expression control machinery. As a result, the presenilin 1 protein in the non-human animal of the invention has the same timing of development, the same tissue specificity and the same synthesis normally associated with the native presenilin 1 protein in the wild-type non-human mammal. Produced with speed.
[0019]
The target transgenic non-human mammal of the invention is used as a tool or model to elucidate the role of PS-1 comprising a human mutation, preferably a human P264L mutation, in AD pathology and symptomology. You may. They may be used to elucidate the manner in which human mutations, preferably the P264L mutation, increase the production of amyloid protein Aβ42. The term "increase" as used herein, when used in the foregoing context, increases the level of Aβ42 produced by a non-human mammal disclosed herein relative to a wild-type control. Means to be done.
[0020]
The non-human mammals and reproductive offspring of the present invention can also be used as assay systems to screen for inhibitors in vivo, and in excess in brain tissue, other tissues and body fluids such as blood, plasma and cerebrospinal fluid. The method may be used to discover and test the potency and suitability of putative compounds for their ability to inhibit the formation, presence and deposition of Aβ peptides in quantity, comprising: (a) native PS-1 A target mutation comprising a human mutation, preferably a human P264L mutation, comprising a sequence encoding a mouse PS-1 peptide containing a human mutation, preferably a P264L mutation, in place of the sequence encoding the peptide. Administering the compound to a non-human mammal homozygous or heterozygous for the mutant PS-1 gene And (b) measuring the amount of Aβ peptide in brain tissue, other tissues and body fluids (or any combination thereof) of the non-human mammal at an appropriate time interval after administration of the compound. Comprising steps.
[0021]
The following terms and phrases as used in this disclosure have the indicated definitions below.
[0022]
As used herein, “Aβ peptide” means either Aβ40 or Aβ42 or a fragment thereof.
[0023]
As used herein, "arm of homology" refers to a site-specific integration of a portion of a targeting vector into a target gene by homologous recombination: (1) homologous recombination. Have sufficient length and homology to provide; (2) one or more mutations to be introduced into the target gene are located therein or therebetween; and (3) positive A nucleotide DNA sequence adjacent to a selectable marker is meant.
[0024]
As used herein, "homologous recombination" refers to the rearrangement of DNA segments at sequence-specific sites or sites within or between DNA molecules by a base-pairing mechanism.
[0025]
As used herein, "a human mutation in the non-human mammalian presenilin 1 (PS-1) FAD gene" refers to the presence of the human PS-1 gene at the corresponding position of nucleotide (s). Any mutation of the PS-1 gene in a non-human mammal that results in the non-human mammal having the corresponding nucleotide (s) is meant. Human mutations in the non-human mammal presenilin 1 (PS-1) FAD gene include, but are not limited to, A79V, V82L, V96F, Y115C, E120D, E120K, M139I, M139T, M139V, I143F, I143T, M146I, M146L (A → T), H163Y, G209V, A231T, A231V, M233T, L235P, L250S, A260V, L262F, C263R, P264L, P267S, R269H, R278E, G280A, E280A , G384A and L392V (each of which is Cruts et al., Human Mutat., 1998, 11, 183-190 (incorporated herein by reference in its entirety). M146L (A → C) (Cruts et al., Human Mutat., 1998, 11, 183-190, Duff et al., Nature, 1996, 383, 710-713, Citron et al., Nature Med., 1997, 3, 67-72, Lee et al., Nature Med., 1997, 3, 756-760 and Lamb et al., Nature Neurosci., 1999, 2, 695- 697, each of which is incorporated herein by reference in its entirety. M146V (Cruts et al., Human Mutat., 1998, 11, 183-190, Duff et al., Nature, 1996, 383, 710-713 and Guo et al., Nature Med., 1999, 5). , H163R (Cruts et al., Human Mutat., 1998, 11, 183-190, Lamb et al., Nature Neurosci., 101-106, each of which is incorporated herein by reference in its entirety). I213T (Cruts et al., 1999, 2, 695- 697 and Chui et al., Nature Med., 1999, 5, 560-564, each of which is incorporated herein by reference in its entirety); Human Mutat., 1998, 11, 183-190 and Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581, each of which is hereby incorporated by reference in its entirety. Shown as); L286V (Cruts et al, Human Mutat. , 1998, 11, 183-190, Citron et al., Nature Med. , 1997, 3, 67-72 and Chui et al., Nature Med. A264E (Cruts et al., Human Mutat., 1998, 11, 183-190, Lee et al.), 1999, 5, 560-564, each of which is incorporated herein by reference in its entirety. Y115H (Nature Med., 1997, 3, 756-760 and Qian et al., Neuron, 1998, 20, 611-617, each of which is incorporated herein by reference in its entirety); Citron et al., Nature Med., 1997, 3, 67-72, which is hereby incorporated by reference in its entirety; T116N (Romero et al., Neuroport., 1999, 10, 2255-2260 ( Exactly the same P117L and L171P (both of which are St. George Hyslop, Biol. Psychiatr., 2000, 47, 183-199, which are incorporated by reference in their entirety). E123L (disclosed in Yasuda et al., Arch. Neurol., 1999, 56, 65-69, which is hereby incorporated by reference in its entirety); N135D, C410Y, A426P and P436S, each of which is disclosed in Hardy et al., Trends Neurosci., 1997, 20, 154-159, which is hereby incorporated by reference in its entirety; Anchin et al., J. Med. Genet., 1998, 35, 672-673 (incorporated herein by reference in its entirety); T147I, W165C, L173W, and S390I, each of which is Campion et al. L166R (Ezquera et al., Arch. Neurol., 2000, 57, Am. J. Human Genet., 1999, 65, 664-670, which is incorporated herein by reference in its entirety); S169L and P436Q, each of which is described in Taddei et al., Neuroport., 1998, 9, 3335-3339 (in its entirety, incorporated herein by reference in its entirety). S169P (Ezquera et al., Neurol.), Incorporated herein by reference in its entirety). E184D (Yasuda et al., Neurosci. Lett., 1997, 232, 29-32 (incorporated by reference in its entirety); E184D (Yasuda et al., Neurosci. Lett., 1997, 232, 29-32). G209R (disclosed in Sugiyama et al., Online Human Mutat., 1999, 14, 90, which is hereby incorporated by reference in its entirety); L219P (Smith et al., Neuroport., 1999, 10, 503-507, which is hereby incorporated by reference in its entirety); M233L and A409T (both of which are Aldudo et al., Human Mut). t., 1999, 14, 433-439 (incorporated herein by reference in its entirety); E273A (Kamimura et al., J. Neurol. Sci., 1998, 160, 76-81 ( L282R (Aldudo et al., Neurosci. Lett., 1998, 240, 174-176, which is hereby incorporated by reference in its entirety); G378A (disclosed in Besancon et al., Human Mutat., 1998, 11, 481, which is hereby incorporated by reference in its entirety); N405S (Yasuda et al., J. Neurol. Neurosurg). .P A409T (Sugiyayama et al., Online Human Mutat., 1999, 14, 90 (cited in its entirety); Sychiatr., 2000, 68, 220-223, which is hereby incorporated by reference in its entirety); L424R (disclosed in Kowalska et al., Folia Neuropath., 1999, 37, 57-61, which is hereby incorporated by reference in its entirety). Δexon 9 splice acceptor site deletion mutation (G → T with S290C) (Hardy et al., Trends Neurosci., 1997, 20, 154-159 and Lee et al., Nature Med. , 1997, 3, 756-760, each of which is incorporated herein by reference in its entirety); a exon 9 splice acceptor site deletion mutation (G → A with S290C). (Sato et al., Human Mutat. Supp., 1998, 1, S91-94, which is hereby incorporated by reference in its entirety); a 4,555 base pair deletion of Exon 9 Finn (Prihar Et al., Nature Med., 1999, 5, 1090, which is hereby incorporated by reference in its entirety); a G deletion of the intron 4 splice donor consensus sequence (DeJonghe et al., Human Molec. Genet). , 1999, 8, 1529-1. 40 (disclosed in its entirety herein by reference)); and a C → T mutation at position −48 in the 5 ′ promoter, a C → G at position −280 in the 5 ′ promoter. Mutations, and the A → G mutation at position −2818 in the 5 ′ promoter (each of which are described in Theuns et al., Human Molec. Genet., 2000, 9, 325-331 (incorporated herein by reference in its entirety). ) Is disclosed. Although the present application particularly exemplifies the P264L mutation, all aspects of the invention can be applied to each and every human mutation cited above.
[0026]
As used herein, a "human P264L mutation" is defined as follows, wherein the nucleotide sequence of codon 264 of the presenilin 1 gene is from the group consisting of CCG: CTT; CTC; CTA; CTG; TTA; TTG. Means to be changed to the selected sequence; and most preferably to be changed from CCG to CTT. In addition, the nucleotide sequence of codon 265 of the presenilin 1 gene is optionally, but preferably, changed from AAA to AAG. The above-described most preferred changes in base sequence in codon 264 constitute the human P264L mutation. Any but preferred changes in the base sequence of codon 265 will add an AflII cleavage site to the gene.
[0027]
As used herein, a "target gene" or "targeted gene" refers to a gene in a cell whose gene is to be modified by homologous recombination using a targeting vector. means.
[0028]
As used herein, a "target transgenic non-human mammal" refers to a non-human mammal comprising one or more target genes via a gene targeting approach as opposed to transgenic. means.
[0029]
As used herein, "progeny by reproduction" with respect to a "target transgenic non-human mammal" encompasses a target transgenic operation whose genome is identical to the parent (s) of the progeny. Means an animal. For example, and without limitation, if a mammal whose genome has been manipulated by gene targeting techniques to encompass human mutations is subsequently used for reproductive purposes, the first mammal (one or more) )), All subsequent generations of origin are referred to as "reproductive offspring" as long as the genome (s) of such subsequent reproductive offspring comprises the target transgenic manipulation as in the original mammal. Considered by design; this definition does not preclude other genomic manipulations that may also be present in such reproductive offspring, and this definition suggests that such reproductive offspring may merely be between male and female mammals. It does not need to be produced only by hybridization techniques.
[0030]
As used herein, a "transgenic non-human mammal" refers to a gene in which a foreign ("transgene") gene sequence has been randomly inserted into the genome of a non-human mammal and is therefore (inserted). A transgene is a non-human mammal that is expressed in addition to all normally expressed native genes (unless the transgene disrupts the gene and thus prevents its expression).
[0031]
As used herein, a "targeting vector" or "replacement vector" refers to a homologous arm, a nucleotide sequence to be incorporated into a target gene (located within or between homologous arms) and one or more. Or a DNA molecule that includes more than one selectable marker.
[0032]
As used herein, a "wild type control animal" is a species of a target transgenic non-human mammal disclosed herein that is otherwise comparable (eg, similar Age) means a non-target transgenic non-human mammal. Wild type control animals can be used as a basis for comparison in assessing results associated with a particular genotype.
[0033]
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
[0034]
The first step in preparing the target transgenic non-human mammal of the present invention is to prepare a DNA targeting vector. The targeting vector is designed to replace a portion of the endogenous presenilin 1 gene sequence of a non-human mammal via homologous recombination so as to introduce a human mutation, preferably a P264L human mutation. The targeting vector is used to transfect non-human mammalian cells, such as pluripotent mouse embryo-derived stem ("ES") cells. In this cell, homologous recombination occurs between the targeting vector and the target gene (ie, a gene targeting event). The mutant cells are then used to generate an intact non-human mammal (eg, by aggregation of mouse ES cells into a mouse embryo) to produce a germline chimera. The germline chimera is used to generate offspring heterozygous for the mutated target gene. Finally, inbreeding of heterozygous offspring results in a non-human mammal (eg, a mouse) homozygous for the mutated target gene.
[0035]
Targeting vectors for the practice of the present invention can be constructed using materials, information and methods known in the art. A general description of the targeting vector used in the present invention follows.
[0036]
The targeting or replacement vector for use in the present invention has two essential functions: (1) specifically (and stably) integrating with the endogenous presenilin 1 target gene; Replacing part of the exon of the presenilin 1 gene, thereby introducing a human mutation and a mutation that introduces a new cleavage site in the gene. In one preferred embodiment, a portion of exon 8 is replaced to introduce a P264L mutation. Their two essential functions depend on two basic structural features of the targeting vector.
[0037]
The first basic structural feature of the targeting vector is a pair of regions known as "homologous arms," which are homologous to selected regions of the endogenous presenilin 1 gene or regions adjacent to the presenilin 1 gene. is there. This homology allows at least a portion of the targeting vector to integrate into the chromosome and replace some (or all) of the presenilin 1 target gene by homologous recombination.
[0038]
Generally, homologous recombination is the rearrangement of DNA segments at sequence-specific sites or sites within or between DNA molecules by a mechanism of base pairing. The present invention relates to one particular form of homologous recombination, sometimes known as "gene targeting".
[0039]
The second basic structural feature of the targeting vector consists of the actual base change (mutation (s)) to be introduced into the target gene. In the present invention, for example, a base change in codon 264 of exon 8 resulted in an amino acid change from proline to leucine at amino acid 264 when the mutated gene was expressed to create a protein. Other base changes can be made as desired to introduce any of the human mutations listed above into the mammalian genome. The mutation (s) to be introduced into the presenilin 1 target gene is located in the "homologous arm".
[0040]
Gene targeting that affects the structure of certain genes already in the cell can be achieved by other forms of stable transformation (integration of exogenous DNA for expression in transformed cells is not site-specific). , And thus do not predictably affect the structure of any particular gene already in the transformed cells). Furthermore, using the types of targeting vectors preferred in the practice of the present invention (eg, those disclosed below), the exchange of genomic DNA (between the “homologous arm” and the target gene) occurs, and the entire vector Chromosome insertion is advantageously avoided.
[0041]
An example of this patent disclosure is the mouse presenilin 1 protein, as it encodes a mouse preserinin 1 protein containing either the human P264L mutation or the other human mutations cited above and an additional cleavage site. 1 shows the construction (and use) of a presenilin 1 gene targeting vector for mutating sequences encoding 1 protein. One skilled in the art will recognize that the principle of homologous recombination can be used to design a number of other targeting vectors to introduce the same mutation. Target transgenic non-human mammals generated using these other targeting vectors are within the scope of the invention. A discussion of targeting vector considerations follows.
[0042]
The length of the homologous arms adjacent to the replacement sequence can vary considerably without significant effect on the practice of the present invention. The homologous arm is of sufficient length for efficient heteroduplex formation between one strand of the targeting vector and one strand of the chromosome of the transfected cell at the locus of the presenilin 1 target gene. Must be something. Increasing the length of the homologous arm promotes heteroduplex formation and thus targeting efficiency. However, it will be appreciated that the increasing targeting efficiency produced by additional homologous base pairs will eventually decrease and be offset by practical difficulties in target vector construction (since homologous arms can exceed thousands of base pairs). Will. The preferred length of each homologous arm is 50 to 10,000 base pairs.
[0043]
There is considerable latitude in selecting which region of the presenilin 1 target gene (ie, the chromosomal region adjacent to the presenilin 1 target gene) is represented in the homologous arm of the targeting vector. The basic constraint is that the base pair to be changed in the presenilin 1 target gene must be within the sequence constituting the homologous arm. Homologous arms may be within the presenilin 1 target gene, but they need not be, and they may be adjacent to the presenilin 1 target gene.
[0044]
Preferably, the targeting vector can include a positive selectable marker between the homologous arms. The positive selectable marker should be located within the intron of the target gene so that it can be spliced from the mRNA and avoid expression of the target / marker fusion protein. More preferably, the targeting vector can include two selectable markers; a positive selectable marker located between the homologous arms and a negative selectable marker located outside the homologous arms. Negative selectable markers are a means of identifying and excluding clones in which the targeting vector has been integrated into the genome by random insertion instead of homologous recombination. Exemplary positive selectable markers are the neomycin phosphotransferase and hygromycin beta phosphotransferase genes. Exemplary negative selectable markers are the Herpes simplex thymidine kinase and diphtheria toxin genes.
[0045]
To eliminate potential interference with target protein expression, a positive selectable marker can be flanked by short loxP recombination sites isolated from bacteriophage P1 DNA. Recombination between the two loxP sites at the locus of the target gene can be induced by introducing an ore recombinase into the cells. This results in the elimination of a positive selectable marker, leaving only one of the two short loxP sites. (See US Pat. No. 4,959,317, which is hereby incorporated by reference in its entirety). Deletion of the positive selectable marker from intron 8 of the mutated presenilin 1 gene can thus be effected.
[0046]
FIG. 1 illustrates the general principle of gene targeting for introducing mutations into the mammalian genome using homologous recombination (Capecchi, MR, Trends Genet., 1989, 5, 70-76; reviewed in Koller and Smithies, Ann. Rec. Immunol., 1992, 10, 705-730). The length of the genomic DNA is first drawn by organizing it into regions (numbered 0-5 in FIG. 1a). In FIG. 1, several base pair changes (from 1 to 10) are to be incorporated into the cellular DNA around region 3. Utilizes homologous recombination using a gene targeting vector. The type of gene targeting vector used to incorporate these changes is termed a replacement vector.
[0047]
As defined above, a "replacement vector" herein includes one or more selectable marker sequences and two contiguous sequences of genomic DNA of an ES cell that flank the selectable marker. Refers to the vector These adjacent sequences are termed "homologous arms". In FIG. 1b, homologous arms are represented by regions 1-2 and 3-4. The use of DNA from ES cells (isogenic DNA) helps ensure high efficiency recombination with the target sequence (te Riele et al., Proc. Natl. Acad. Sci. USA, 1992, 89). , 5128-5132). The homologous arms are located on either side of the DNA sequence encoding the resistance to drugs toxic to ES cells (positive selectable marker) in the vector. The gene encoding sensitivity to another non-toxic drug (negative selectable marker) is located outside the region of homology. In the replacement vector used in the present invention, the positive selection marker is neo^r(Gene encoding resistance to the neomycin analog G418), and the negative selectable marker is the herpes simplex virus thymidine kinase gene (HSV-tk), which encodes sensitivity to ganciclovir. If this replacement vector is introduced into the ES cells via transfection and the DNA undergoes homologous recombination with the DNA of the ES cells, a positive selectable marker in this example is between region 2 and 3 While being inserted into the genome (making the transformed cells resistant to G418), the negative selectable marker is eliminated (making the cells resistant to ganciclovir). Therefore, to enhance for homologous recombination, transfected ES cells were^rGrow in a medium containing G418 to select for the presence of the gene and ganciclovir to select for the absence of the HSV-TK gene. Preferably, the positive selectable marker is isolated once the mammal has been bred to another mammal, for example, using cre / lox technology.
[0048]
If base pair changes (mutations) are introduced into one of the homologous arms, these changes can be integrated into the cell's gene as a result of homologous recombination. Whether a mutation is integrated into cellular DNA as a result of homologous recombination depends on where the crossover event occurs in the homologous arm carrying the change. For example, as depicted by scenario "A" in FIG. 1, crossovers in the arms occur proximal to the mutation, and thus they do not integrate into cellular DNA. In scenario "B", the crossovers occur distal to the location of the mutation and they are integrated into the cellular DNA. Since the location of crossover events is random, the frequency of homologous recombination events, including mutations, is increased when they are placed closer to a positive selectable marker.
[0049]
The above method allows one skilled in the art to achieve incorporation of a selectable marker at a preselected position in the gene of interest flanked by particular base pair changes. Possibly, those skilled in the art will preferably choose to have the selectable marker incorporated within an intron of the gene of interest so as not to interfere with endogenous gene expression, while retaining the desired selectivity in the protein product of interest. It appears that mutations are included in adjacent coding sequences to make the change (FIG. 1), (Askew et al., Mol. Cell. Biol., 1993, 13, 4115-4124, Fiering et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8469-8473; Rubinstein et al., Nuc. Acid. Res., 1993, 21, 2613-2617, Gu et al., Cell, 1993, 73, 1155-1164 and Gu et al. Science, 1994, 265, 103-106)
[0050]
Accordingly, a target transgenic non-human mammal comprising a human PS-1 mutation is prepared in the manner described above. The mammal can be heterozygous (containing one copy of the human PS-1 mutation) or homozygous (containing two copies of the human PS-1 mutation). In one preferred embodiment, PS-1^{P264L / +}(Heterozygous) or PS-1^{P264L / P264L}(Homozygous) mice are prepared.
[0051]
A target transgenic non-human mammal comprising a human PS-1 mutation as described above is substituted for a mammal having a Swedish FAD mutation in the APP gene and a "humanized" Aβ sequence (eg, APP).^{NLh / NLh}Mouse) and APP^{NLh / NLh}× PS-1^{P264L / P264L}, APP^{NLh / +}× PS-1^{P264L / P264L}, APP^{NLh / +}× PS-1^{P264L / +}Or APP^{NLh / NLh}× PS-1^{P264L / +}Mammals can be produced. In addition, a target transgenic non-human mammal comprising a human PS-1 mutation as described above can be bred to a mammal having a Swedish APP695 mutation (eg, a Tg2576 mouse). However, prior to mating such mammals, neo / neo using cre / lox technology^rIt is preferable to remove a positive selection marker such as
[0052]
The present invention is also directed to a method for identifying a compound for treating Alzheimer's disease. The compound may be heterozygous or homozygous for a mutation in the PS-1 gene and also contain a human Swedish APP695 mutation or its reproductive offspring, or a mutation in the PS-1 gene, human FAD It is administered to mammals heterozygous or homozygous for the Swedish mutation and the humanized Aβ mutation and their reproductive offspring. Any compound to be tested can be administered in various amounts by any of a variety of routes, including but not limited to intravenous, oral, direct infusion into the brain, and the like. A tissue sample from a mammal, including but not limited to brain tissue, non-brain tissue and body fluids (eg, blood and plasma) is obtained, and the amount of Aβ peptide in the tissue sample is measured. A decrease in the amount of Aβ peptide in a tissue sample is indicative of a compound that can be used to treat Alzheimer's disease.
[0053]
The invention is also directed to a method of treating an individual suspected of having Alzheimer's disease. An individual suspected of having Alzheimer's disease is any human who has been examined by a physician and diagnosed as having Alzheimer's disease or symptoms thereof. A mammal heterozygous or homozygous for a mutation in the PS-1 gene and also containing a human Swedish APP695 mutation or a reproductive offspring, or a mutation in the PS-1 gene, a human FAD sudden mutation in Swedish Compounds identified by the methods described above for mammals that are heterozygous or homozygous for the mutation and the humanized Aβ mutation, as well as for their reproductive offspring, reduce the amount of Aβ peptide in the brain of the individual. Administered to the individual in an effective amount. An amount effective to reduce the amount of Aβ peptide is, as a starting amount, a mammal or a mammal thereof that is heterozygous or homozygous for the mutation of the PS-1 gene and that also contains the human Swedish APP695 mutation. Using a reproductive offspring or a mammal or a reproductive offspring that is heterozygous or homozygous for the mutation of the PS-1 gene, the human FAD Swedish mutation and the humanized Aβ mutation And, as is known to those skilled in the art, can be determined from methods of identifying such compounds, which increase with rate for use in humans. An effective Alzheimer's disease therapeutic amount is an amount of a compound that measurably reduces the physiological condition of Alzheimer's disease, or an amount that reduces the physical manifestation or signs of Alzheimer's disease. One skilled in the art can, for example, start with an amount of a compound that reduces the amount of Aβ peptide in the brain, as described above, and depending on the desired effect and the effect achieved in a particular individual, The amount can be increased or decreased depending on the rate.
[0054]
The present invention is also directed to compounds identified by the screening methods described above. The compound can be any identifiable chemical or molecule, including but not limited to small molecules, peptides, proteins, sugars, nucleotides or nucleic acids, and such compounds can be natural or synthetic. Can be.
[0055]
Examples are provided below to provide a more efficient understanding of the invention disclosed herein. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting to the invention in any manner. Throughout these examples, molecular cloning reactions and other standard recombinant DNA techniques were performed using commercially available enzymes, as described in Maniatis et al., Molecular Cloning-A Laboratory Manual, unless otherwise indicated. , 2nd edition, Cold Spring Harbor Press (1989) (below, "Maniatis et al.").
[0056]
【Example】
Example 1: Cloning of mouse PS-1 exon 8 region
Mouse PS-1 genomic DNA was obtained from Lambda DASH from a bacteriophage library made from 129 / Sv mouse DNA partially digested with Sau3A.^TM  II was cloned into the BamHI site (Reaume et al., Science, 1995, 267, 1831-1833, which is incorporated herein by reference in its entirety). Using standard molecular biology techniques (Maniatis et al.), Hybridization with a small radiolabeled PS-1 specific DNA probe yields approximately 1.2 × 10⁶Individual recombinant bacteriophages were screened for the presence of the PS-1 sequence. The 477 base pair PS-1 probe was prepared using primers R892 (CTC ATC TTG GCT GTG ATT TCA; SEQ ID NO: 1) and R885 (GTT) which hybridize to the 3 ′ end of exon 7 and the 5 ′ end of exon 11, respectively. Generated by polymerase chain reaction (PCR) amplification of mouse genomic DNA using GTG TTC CAG TCT CCA; SEQ ID NO: 2) (Mullis et al., Methods Enzymol., 1987, 155, 335-350) (Figure). 2). The amplified fragment was separated from other reaction components by electrophoresis on a 1.0% agarose gel and purified using GeneClean ™ II (Bio 101, Inc., La Jolla, Calif.). Purified probe DNA was obtained by random primer method using materials and methods (Multiprime DNA Labeling System; Amersham Life Sciences, Arlington Heights, IL) supplied by the kit manufacturer.³²Radiolabeled with P-dCTP.
[0057]
From this screen, 13 clones that hybridized to the PS-1 probe were identified. Clones were identified as follows: λPS1-4, λPS1-5, λPS1-6, λPS1-10, λPS1-11, λPS1-17, λPS1-19, λPS1-20, λPS1-22, λPS1-24. λPS1-28, λPS1-31, and λPS1-35. These clones were purified by limiting dilution and plaque hybridization with PS-1 probe (Maniatis et al.).
[0058]
From each clone, DNA was prepared from bacteriophage particles purified on a CsCl gradient (Maniatis et al.). Then, for each of the cloned inserts, FLASH^TM  Restriction enzyme maps were generated using the Non-radioactive Gene Mapping Kit (Stratagene ™ Inc., La Jolla, CA). A typical restriction map generated by this method is illustrated in FIG. This method of restriction enzyme mapping involves first completely digesting 10 μg of bacteriophage DNA with the restriction enzyme NotI using standard restriction enzyme digestion conditions (Maniatis et al.). . NotI was cloned from all clones in the vector DNA to separate the T3 bacteriophage promoter attached to one end of the insert and the T7 bacteriophage promoter attached to the other end. Cut at both ends of the inserted insert. The NotI digested DNA is then partially digested with the enzyme EcoRI using a limited amount of enzyme (0.2 units / μg DNA) in an 84 μl reaction volume at 37 ° C., as an example. . After 3, 12, and 40 minutes, aliquots (26 μl) were removed and the digestion reaction was stopped by the addition of 1 μl 0.5 M EDTA. DNA from all three time points was separated on a 0.7% agarose gel, visualized by ethidium bromide staining, and then, by capillary transfer, a GeneScreen Plus ™ membrane (NEN ™ Research Products, Boston, Mass.). (Maniatis et al., Supra). This membrane was prepared using a reagent and method supplied by the kit manufacturer, using an alkaline phosphatase-labeled oligonucleotide (FLASH) specific for the T3 promoter.^TM(Included in the kit). After hybridization, the membrane was washed, developed with a chemiluminescence producing substrate, and then exposed to X-ray film for about 60 minutes in the dark.
[0059]
The oligonucleotide probe effectively labels one end of the insert. By determining the position of the bands on the X-ray film and calculating the size of the DNA to which they corresponded, it was possible to determine the position of the EcoRI site relative to the T3 end of the insert (FIG. 3). These results could then be supplemented by stripping the probe from the membrane and rehybridizing a T7 specific oligonucleotide to determine the location of the EcoRI site relative to the T7 end of the insert. This process was repeated with the enzymes HindIII and XbaI. A synthetic map was constructed by comparing the restriction maps of the different clones that overlapped. From the first 13 clones isolated, six independent clones were identified (FIG. 2).
[0060]
Exon 8 was located on the restriction map by hybridizing an exon-specific probe to the complete digest of each of the six different lambda genomic clones. Initially, 3 μg of DNA from each of the 6 different clones was completely digested with the restriction enzymes EcoRI and XbaI. The digested DNA was resolved on a 0.8% agarose gel, visualized by ethidium bromide staining, and transferred to a GeneScreen Plus ™ membrane by capillary transfer. This membrane was then hybridized with a DNA probe that specifically hybridized to sequences from mouse PS1 exon 8. This probe comprises oligonucleotides FEX8 (ATT TAG TGG CTG TTT TGT G; SEQ ID NO: 3) and REX8 (AGG AGT AAA TGA GAG CTG GA; SEQ ID) which hybridize to the 5 'end and 3' end of exon 8, respectively. NO: 4). After hybridization, the membrane was washed and exposed to X-ray film (FIG. 4). This experiment revealed that all clones contained a 2.5 kb fragment that hybridized to the exon 8 probe. By combining this information with the restriction map data for each lambda clone, exon 8 was identified on the map (positions 11.5 to 14, on the summary map, FIG. 2).
[0061]
A similar procedure was used to identify the location of exon 7 on our synthetic map using exon 7 specific probes and utilizing the restriction enzymes XbaI and EcoRI. An exon 7-specific probe was prepared using PCR primer F892 (TGA AAT CAC AGC CAA GAT GAG; SEQ ID NO: 5) and PS1-1 (GCACTCC CTG ATC TGG AAT TTT G; SEQ ID NO: 6). Was. Exon 7 is localized to the 2 kb XbaI-EcoRI fragment of all clones except λPS1-22, which allows the determination that exon 7 is located between positions 7.0 and 9.0 on the summary map. (Fig. 2).
[0062]
Exon-specific probes were also used to obtain additional restriction map information using additional restriction enzymes. For example, λPS1-22 was digested with NotI and BamHI, separated on an agarose gel, transferred to a Genescreen Plus ™ membrane, and probed with an exon 8 specific probe, which identified a 700 bp fragment. This information, when combined with information from other bacteriophage clones, allowed placement of BamHI at position 11.7 on the synthetic map (FIG. 2). This process was repeated for the restriction enzyme XhoI.
[0063]
Also, cloning of additional regions of the mouse PS-1 gene to prepare additional vectors containing other human mutations can be accomplished as desired.
Example 2: Construction of replacement vector
A 4.7 kb Xbal-BamHI fragment (including two internal Xbal fragments) located at positions 7.0 to 11.7 on the summary map (Figure 2) was selected as the 5 'arm containing the required mutation. For the 3 'arm, a 4.1 kb BamHI-EcoRI fragment (including an internal EcoRI site) located at positions 11.7 to 15.8 on the summary map (FIG. 2) was selected. These fragments were first isolated and generated with pBlueScript ™ SK + (Stratagene Cloning Systems, LaJolla, LA) to generate a replacement vector (pPS1-8-TV, FIG. 5) having the same general structure as described above. CA) and then neo^rGene, HSV-TK gene and pPNTIox containing linker sequence²It was further subcloned into a plasmid (described below).
[0064]
(A) Intermediate plasmid pPNTlox²Production of
pPS1-8-TV was initially neo^rInsert two oligonucleotide linkers on each side of the cassette and insert pPNTIox²It was made from pPNT (Tyblewicz et al., Cell, 1991, 65, 1153-1163; obtained from MIT Dr. Richard Mulligan) by making an intermediate called (FIG. 6). The double-stranded 79 base pair 5 'linker allows the two single-stranded oligonucleotides overlapping at the 3' end to anneal and then fill the remaining single-stranded region with the Klenow fragment of DNA polymerase I. Was produced. Oligonucleotide PNT Not (GGA AAG AAT GCG GCC GCT GTC GAC GTT AAC ATG CAT ATA ACT TCG TAT; SEQ ID NO: 7) ID NO: 8) (150 ng each) was bound in a 30 μl reaction mixture containing 5 U of Klenow polymerase, Klenow polymerase buffer and 2 mM dNTPs (dATP, dCTP, dGTP and dTTP). After incubation at 37 ° C. for 1 hour, a portion (5 μl) of this reaction mixture was digested simultaneously with restriction enzymes NotI and XhoI to release restriction enzyme sites at each end of the linker. In addition, 200 ng of pPNT was digested with NotI and XhoI. The digested plasmid was separated on a 0.8% agarose gel, purified from the gel, and treated with calf intestinal phosphatase according to standard methods (Maniatis et al.). An aliquot (66 ng) of the double digested linker was ligated to the double digested and phosphatase treated pPNT DNA (Maniatis et al.). After DNA transformation of competent WM 1100 E. coli (Dower, Nucleic Acids Res., 1988, 16, 6127-6145), plasmid DNA was isolated from ampicillin resistant bacteria (Holmes et al., Anal. Biochem., 1981, 114, 193-197) and analyzed by restriction analysis. A suitable recombinant plasmid was identified as having acquired the SalI, HpaI and NsiI sites (present in the linker) while still retaining the NotI and XhoI sites of the starting plasmid. One such recombinant plasmid with a 79 bp linker sequence was identified and called pXN-4 (FIG. 6).
[0065]
A similar approach was used to insert a 3 'linker between the XbaI and BamHI sites of pXN-4. Oligonucleotides used for synthesizing the linker were PNT Xba (CGT TCT AGA ATA ACT TCG TAT AAT GTA TGC TAT; SEQ ID NO: 9) and PNT Bam (CGT GGA TCC ATA ACT TAT CATA GAT TAT GAT TAT CATA GAT SEQ ID NO: 10). In this case, pXN-4 and double-stranded linker DNA were digested with XbaI and BamHI. The purified fragments were ligated by DNA ligation and transformed into competent WM1100 bacteria. Plasmid DNA was digested with XbaI and BamHI,³²End-labeled with P-dCTP and Klenow polymerase and separated on an 8% acrylamide gel (Maniatis et al.). The gel was dried and exposed to X-ray film. Suitable recombinant clones were identified by the presence of a 40 base pair band released by XbaI-BamHI double digestion. The resulting plasmid is called "pPNTIox".²"(FIG. 6).
[0066]
To verify the sequence of the inserted linker, a fragment containing both linkers was cloned into pPNTIox using NotI and EcoRl.²And cloned into pBlueScript ™ SK +, a vector more amenable to nucleotide sequencing. Linker identity was determined using T3 and T7 sequencing primers (Stratagene ™ Inc., La Jolla, Calif.) And the Sequenase Version 2.0 DNA Sequencing Kit (using the United States Biochemical, directly using United States Biochemical, Cleveland). Nucleotide sequencing (Sanger, Proc. Natl. Acad. Sci. USA, 1977, 74, 5463-5467).
[0067]
(B) Subcloning of homology arms.
[0068]
The XbaI-HindIII fragment (positions 11.5 to 15.9 on the summary map, FIG. 2) containing the homologous 3 'arm and the fragment used for in vitro mutagenesis, initially contained 30 μl of phage DNA. Digest with XbaI and HindIII, separate the digested DNA on a 0.8 agarose gel, visualize the DNA with ethidium bromide staining, and then excise the λPS1-22 by excising the 4.4 kb fragment from the gel. Released. DNA was purified from the gel using GeneClean ™ II (Bio 101 Inc., La Jolla, Calif.). At the same time 1 μg of pBlueScript ™ SK + was digested with XbaI and HindIII and subsequently purified by the same procedure. Approximately 400 ng of purified lambda DNA and 100 ng of purified plasmid DNA were ligated in a lOμl ligation reaction. After transformation of competent WM 100 E. coli, plasmid DNA was isolated from ampicillin-resistant bacteria and analyzed by restriction enzyme analysis to confirm the resulting plasmid (FIG. 7). In this case, plasmid DNA from the transformed bacteria was analyzed by first digesting it with XbaI and HindIII to determine if the plasmid DNA had acquired the 4.4 kb PS-1 fragment. . This plasmid was named "pPS I-XH16".
[0069]
A similar procedure was used to isolate a 200 bp XbaI-BamHI fragment from pPS1-XH16 and subclone it into pBlueScript ™ SK +. The resulting plasmid was named "pPS1-XB1" (FIG. 8).
[0070]
One of the fragments in the homologous 5 'arm (4.2 kb XbaI fragment from positions 7.0 to 11.2 on the summary map; FIG. 2) also contained within λPS1-6 to pBlueScript ™ SK +. It was subcloned and named "pPS1-X15" (FIG. 9). Since the insert could be placed in the plasmid in either of two orientations, the plasmid DNA was further screened by digesting it with the enzyme EcoRI. Thus, clone pPS1-X15 has a PS-1 insert with the 5 'end closest to the T3 promoter, while the 5' end in pPS1-X2 is adjacent to the T7 promoter. Was determined (FIG. 9).
[0071]
The 300 bp XbaI fragment in the 5 'arm (positions 11.2 to 11.5 on the summary map; FIG. 2) was also cloned similarly from λPS1-20 into pBlueScript ™ SK +, and pPS1-X319 (FIG. 10). In this case, the orientation of the XbaI fragment was not determined by subsequent restriction mapping.
[0072]
(C) Restriction enzyme mapping of homology arms.
[0073]
Further restriction enzyme mapping was performed on pPS1-X315 and pPS1-X2. As an example, each of the two plasmids was digested with the enzyme HincII, separated on an agarose gel and visualized with ethidium bromide. In each of the two digested samples, a fragment was identified in each of the two digested samples, since a HincII site is known to be present in the pBlueStrip ™ SK + plasmid backbone within the multiple cloning site region close to the T7 promoter as compared to the position of the insert. By determining the size of HincII, it was possible to determine the location of the HincII site in the 4.2 PS-1 fragment (FIG. 11).
[0074]
The location of the restriction site once or twice in the 4.2 kb PS-1 fragment was determined by the method described above. If there are more than two sites for a given enzyme, each of the two plasmids is double digested with the enzyme in question as well as another enzyme that cuts a site that can resolve ambiguity. By doing so, it became necessary to determine the relative position. In many cases, enzymes that cleave more than twice were not resolved in this way and were simply described as having multiple sites in the 4.2 kb PS-1 fragment. A list of other enzymes used to characterize this region includes, but is not limited to, AccI, ApaI, BamHI, EcoRI, HincII, HpaI, KpnI, NsiI, PstI, SalI, SmaI, SpeI and XhoI. . A summary of these data is illustrated in FIG. The same procedure was used to generate a restriction map of pPS1-XH16 (FIG. 12).
[0075]
(D) Mutation of homologous 3 'arms.
[0076]
A total of 3 base pair changes were introduced into the exon 8 region using a PCR strategy (see FIG. 13 for a summary of the changes). The P264L mutation and an AflII site were introduced. 10 ng of pPS1-XB1 was included in each of the two PCR reactions. The first reaction included primers EXPL2 (TTG TGT CTT AAG GGT CCG CTT CGT ATG; SEQ ID NO: 11) and T7 (Stratagene Cloning Systems, La Jolla, CA). This generated a 220 bp band encompassing the 3 'end of exon 8 and clone PS1-XB1. This fragment also contained the P264L mutation and a new AflII site created as part of the P264L mutation.
[0077]
The second PCR reaction used primers EXPL1 (CGG ACC CTT AAG ACA CAA AAC AGC CAC; SEQ ID NO: 12) and T3 (Stratagene Cloning Systems, La Jolla, CA). This generated a 137 bp fragment encompassing the 5 'end of exon 8 and clone PS1-XB1. This fragment also contained the P264L mutation and an AflII site (FIG. 14).
[0078]
The product of the first reaction is Magic^TM  Purified using the PCR Preps DNA Purification System (Promega Corporation, Madison, Wis.) And digested with BamHI and AflII to release the terminal restriction sites. Similarly, the product of the second reaction was purified and digested with AflII and XbaI. These two fragments and pBlueScript ™ SK + digested with XbaI and BamHI were ligated together and transformed into HB101 competent E. coli cells. DNA was isolated from ampicillin resistant colonies and analyzed. The clone carrying the recombinant plasmid in which the two PCR fragments were ligated together at their AflII sites and inserted into the BamHI and XbaI sites of pBlueScript ™ SK + was called pPS1-XB85 (FIG. 14). . To confirm the sequence of the mutagenized exon 8, T3 and T7 sequencing primers (Stratagene Inc., LaJolla, Calif.) And Sequence Version 2.0 DNA Sequencing Kit (United States Biochemical, using United States Biochemical, USA) Direct nucleotide sequencing (Sanger, Proc. Natl. Acad. Sci. USA, 1977, 74, 5463-5467) was performed.
[0079]
The homologous 5 'arm was assembled into pBlueScript ™ SK + through several cloning steps. First, pPS1-XB15 was partially digested with XbaI such that only one XbaI site was cut. The resulting DNA was then digested with BamHI and gel purified (FIG. 15).
[0080]
The mutated insert in pPS1-XB85 was released by digesting it with XbaI and BamHI and gel-purifying the resulting mutated insert. A 200 bp XbaI-BamHI fragment was ligated into the linearized pPS1-X15 and the recombinant plasmid was screened by AflII digestion for proper orientation of the insert. Plasmids with the correct orientation yielded 1.9 kb and 5.8 kb fragments. This plasmid was named "pPS1-206".
[0081]
To insert the small 300 bp XbaI fragment 5 'to the mutated 200 bp XbaI-BamHI fragment, pPS1-206 was linearized by partial XbaI digestion (FIG. 16). The XbaI fragment from pPS1-X319 was isolated and cloned into linearized pPS1-206 DNA. The orientation of the 300 bp XbaI fragment was determined by sequencing the recombinant clone and λPS1-20 with the EX8PL1 primer using a Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Life Science Inc., Cleveland, OH). Determined by One plasmid clone that shared sequence identity with λPS1-20 across the Xbal site had the 300 bp Xbal fragment inserted in the proper orientation. This plasmid containing the assembled 5 'arm was named "pPS1-5'360" (Figure 16).
[0082]
(E) Assembly of the targeting vector pPS-1-8-TV.
[0083]
Plasmid pPNTlox²Was prepared by first digesting plasmid DNA with EcoRI and BamHI to accept the homologous 3 'arm and gel separating the linear plasmid (Figure 17). In parallel, the 3 'arm was prepared by partially digesting pPS1-XH16 with EcoRI and isolating the linear form. This fragment was then digested with BamHI and the 4.1 bp fragment was separated by gel. 3 'arm is pPNTlox²Was linked to. The resulting plasmid was named "pPNT3'413".
[0084]
The 5 'arm was inserted into pPNT3'413 to give the final pPSI-8-TV plasmid. The 5 'arm was released from the plasmid DNA by first digesting with XhoI and NotI. In parallel, pPNT3'413 was prepared by double digestion with NotI and SalI. The two DNA fragments were ligated and transformed into competent WM 1100 E. coli cells (FIG. 18).
[0085]
Additional vectors can be prepared in the above method to include other human mutations.
Example 3: Mutagenesis of mouse PS-1 gene in ES cells
(A) Cells.
[0086]
129 / Sv × 129 / Sv-CP FI hybrid mice (Nagy et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8424-8429), Dr. Janet Rossant (Mt. Sinai Hospital, T.R.). , Ontario, Canada) were used. These cells contain 20% fetal calf serum (FBS; Hyclone Laboratories Inc., Logan, Utah; cat. # A-1115; Lot # 11152154), 0.1 mM non-essential amino acids (Mediatech 25-025-L1), 2 mM. L-glutamine (Mediatech 25-005-L1), 10^-6M β-mercaptoethanol (Gibco 21985-023), 1 mM sodium pyruvate (Mediatech 25-000-L1), 1 × concentration of penicillin-streptomycin solution (Mediatech 30-001-L1) and 1000 U / ml leukemia inhibitory factor ( Cultures were performed in ES cell medium (containing L-glutamine and 4500 mg / L glucose; Mediatech Inc., Herndon, VA), supplemented with Dulbecco's modified Eagle's medium, supplemented with Gibco BRL 13275-029). Cells were cultured on tissue culture plastic that had been briefly treated with a 0.1% gelatin (Sigma G9391) solution.
[0087]
Cultures were passaged every 48 hours or when cells were approximately 80% confluent. For passage, cells are first in phosphate buffered saline (Ca²⁺And Mg²⁺), And then trypsin / EDTA solution (Ca²⁺And Mg²⁺(0.05% trypsin and 0.02% EDTA in PBS) containing no. After all of the cells were suspended, tryptic digestion was stopped by the addition of tissue culture medium. Cells were harvested by centrifugation, resuspended in 5 ml of tissue culture medium, and a 1 ml volume of cell suspension was used to start a new plate of the same size.
[0088]
(B) DNA transfection of ES cells.
[0089]
pPS1-8-TV DNA (400 μg) was prepared for electroporation by digesting it with NotI in a 1 ml reaction volume. The DNA was then precipitated by the addition of ethanol, washed with 70% ethanol and resuspended in 500 μl of sterile water.
[0090]
PPS1-8-TV DNA linearized with NotI was electroporated into ES cells using a Bio-Rad Gene Pulser ™ System (Bio-Rad Laboratories, Hercules, CA). In each of the ten electroporation cuvettes, 2.5 × 10 5 suspended in ES cell culture medium⁶Individual cells were electroporated with 40 μg of DNA. Electroporation conditions were 250 V, 500 μF, which typically resulted in a time constant ranging between 6.0 and 6.1 seconds. After electroporation, cells were incubated for 20 minutes at room temperature in an electroporation cuvette. All electroporated cells were then pooled and distributed equally on 10 gelatinized plates. After 24 hours, the plates were aspirated and fresh ES cell medium was added. The next day, the media in the nine plates was replaced with ES cell media supplemented with 150 μg / ml G418 (Gibco) and 0.2 μM ganciclovir (Syntex), while one plate contained only 150 μg / ml G418 Medium supplemented with. After an additional 8 days, the resulting individual ES cell colonies were picked from the plates and described by Wurst et al. , Pp 33-61 in Gene Targeting Vol. 126, 1993, Edited by A. L. Grow individually in wells of a 24-well plate as described by Joyner, IRL Press, Oxford University Press, Oxford, England. Comparison of the number of colonies grown on plates supplemented with G418 and ganciclovir versus the number grown only with G418 supplementation was used to determine the efficiency of the negative selection.
[0091]
(C) Analysis of ES cell transformants.
[0092]
When the cell culture in each well of the 24-well plate was about 80% confluent, it was washed and the cells were sprayed with 2 drops of trypsin-EDTA. Trypsinization was stopped by the addition of 1 ml of ES cell medium. An aliquot (0.5 ml) of this suspension was transferred to each of two wells of a separate 24-well plate. After the cells had grown to near confluence, one of the plates was used for cryopreservation of the cell line, while the other was used as a source of DNA for each of the clones.
[0093]
For cryopreservation, cells in 24-well plates were first chilled by placing the plates on ice. The medium was then replaced with fresh ES cell medium supplemented with 10% DMSO and 25% FBS, and the plates were insulated in a styrofoam box by placing them in a -70 ° C freezer, approximately It was cooled at 0.5 ° C./min.
[0094]
To isolate DNA from clones on other plates, medium in each well was supplemented with 500 μl digestion buffer (100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 μg / ml proteinase K). After overnight incubation at 37 ° C., 500 μl of isopropanol was added to each well and the plates were agitated on an orbital shaker for 15 minutes. The supernatant was aspirated, replaced with 500 μl of 70% ethanol, and the plate was shaken for another 15 minutes. The DNA precipitate was removed from the wells and dissolved in 50 μl of TE solution (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).
[0095]
Primary analysis for mutagenesis of the mouse PS-1 gene involved a Southern hybridization screen of ApaI digested ES cell DNA. Probes for this analysis were derived from the 3 'end of our cloned PS-1 region outside the homologous 3' arm (Figure 19d). It was prepared by first isolating a 6 kb XbaI fragment corresponding to the 3 'end of? PS1-6 (Figure 2) and subcloning it into XbaI digested pBlueScript (TM) SIB +. Further digestion of this subclone, called pPS1-X6, with XhoI (internal site) and HindIII (from Bluescript ™ SK + polylinker) resulted in a 1000 bp probe.
[0096]
For Southern hybridization screening, an aliquot (10 ul) of each ES cell clone DNA was digested with ApaI, separated on a 0.8% agarose gel, and transferred to a GeneScreen Plus ™ membrane. Probes are random primed³²Labeled with P-dCTP and hybridized to the membrane at 58 ° C. overnight (Church et al., Proc. Nad. Acad Sci, USA, 1984, 81, 1991-1995). ES cell lines in which the PS-1 gene has successfully undergone homologous recombination yield 9 and 15 kb ApaI fragments by this assay (FIG. 19). This is because homologous recombination is convenient and a new ApaI site is added to neo.^rThis is because the cassette is introduced into an area where the cassette is taken in. The 15 kb band represents an unaltered cell copy of PS-1, while the 9 kb band is derived from the PS-1 copy where a new ApaI site results in a shorter fragment. Eight of the 260 cell lines analyzed in this initial screen were identified as potential successful targeting cell lines.
[0097]
All cell lines evaluated by the primary screen as putative homologous recombinants were then purified using the 500 bp KpnI-ApaI fragment isolated from the 5.5 kb 5 ′ XbaI fragment from λPS1-20. Further screening was performed on ScaI digested ES cell DNA. In this case, the normal PS-1 gene yielded a 13.8 kb fragment and the mutant PS-1 gene yielded a 10.5 kb fragment (FIG. 19e). Four out of the eight cell lines examined by this screen showed that they had undergone homologous recombination at their 5 'end.
[0098]
Cell lines identified as having undergone homologous recombination by both screens were identified as having undergone truly homologous recombination (for homologous insertions that gave a positive result in only one of the two previously described screens). Was considered. However, the mutations included in this arm may or may not be incorporated into cellular DNA as a result of appropriate homologous recombination, depending on where the cross-over occurs when the 5 'arm undergoes recombination. May not be possible (Fig. 1). Therefore, an additional Southern hybridization screen aimed at detecting new AflII sites resulting from the P264L mutation was performed. To this end, a 1.2 kb HindIII-XbaI fragment isolated from pPS1-X15 was used as a probe for AflII-digested DNA. The unaltered PS-1 gene yielded a 6.7 kb band (FIG. 19f). Appropriate homologous recombination has occurred, but the PS-1 gene lacking the planned mutation produces a 8.7 kb band, while the inclusion of the planned mutation produces a 2.2 kb band. . Of the four true homologous recombination cell lines examined, all four were shown to have incorporated new AflII sites near the proposed mutation.
[0099]
The mutated form of the PS-1 gene described herein is a PS-1 gene.^nP264L, Whereas the normal PS-1 gene is PSI⁺It was named. One copy of PS-1^nP264LThe four ES cell lines that harbored PS1 were designated PS1-87, PS1-175, PS1-176 and PS1-243. Three of these strains were thawed, cell numbers expanded, and used to establish PS-1 mutant mice.
[0100]
Further mutagenesis of the mouse PS-1 gene can be performed in ES cells in the above method to include other human mutations.
Example 4: Establishment of PS-1 mutant mouse
PS-1 mutant ES cells were used to generate chimeric mice by aggregating mutant ES cells into E2.5 embryos and transferring the aggregated embryos to pseudopregnant females (Wood et al., Nature, 1993, 365, 87-89). ES cells were prepared for aggregation by limited trypsinization to produce aggregates that averaged 10-15 cells. E2.5 embryos were obtained from Hogan et al. , Manipulating the Mouse Embryo: A Laboratory Manual, 1986, Cold Spring Harbor Laboratory, Cold Spring Harbor, Cold Spring Harbor, New York, USA. Recovered. Zona pellucida was removed from embryos using acidic Tyrode's solution (Sigma Chemical Co., St. Louis, MO). Aggregation wells were created by pushing a smooth metal instrument (overhead) into tissue culture plastic. Embryos were then placed in wells with approximately 10-15 ES cell clumps in M16 medium (Sigma Chemical Co., St. Louis, MO) in droplets (about 20 μl) under mineral oil. Was cut. Overnight incubation (37 ° C, 100% humidity, 5% CO in air)₂After) the aggregated embryos were transferred to the uterine horn of pseudopregnant females (Hogan et al., 1986, supra).
[0101]
The contribution of ES cells to progeny was assessed by the appearance of pigmented coat color. Positive mice are called chimeric founders. Germline contribution by ES cells was assessed by the appearance of colored progeny from the cross between the chimeric founder and the CD-1 female.
Example 5: Chimera
Of the three PS-1 mutant ES cell lines used for embryo aggregation, one produced a germline chimera:
[0102]
[Table 1]

[0103]
The germline chimera was then PS-1^nP264LWas used to establish strains of mice carrying The presence of the mutant PS-1 allele in the colored offspring follows substantially the same procedure as described in Example 1, followed by neo^rIt was determined using a PCR strategy aimed at detecting the cassette. The PCR primers were as follows: neo28 (GGA TTG CAC GCA GGT TCT CC; SEQ ID NO: 13); and neo445 (CCG GCT TCC ATC CGA GTA CG; SEQ ID NO: 14). Genomic DNA was prepared from tail samples (Hogan, 1986, supra). Of the four colored offspring, one female mouse had PS-1^nP264LFor heterozygosity (PS-1^{nP264L / +})Met. That is, this mouse is neo-based on the aforementioned PCR strategy.^rPositive for cassette. Similarly, PS-1^nP264LSubsequent offspring that are heterozygous for were developed by crossing this female with a wild-type male.
[0104]
PS-1^nP264LFor heterozygosity (PS-1^{nP264L / +}) Were determined for genetic traits using a PCR-based method. The presence of the wild-type allele of mouse PS-1 was evaluated using the following primers: X8F (CCC GTG GAG GAG GTC AGA AGT CAG; SEQ ID NO: 15) and X8R (TTA CGG GTT GAG CCA TGA ATG SEQ ID NO: 16). Evaluation with these primers yields a 142 bp fragment (data not shown). The presence of the mutant allele was assessed using primers neo28 and neo445, which yielded a 417 bp fragment. Thus, mice heterozygous for the mutation give rise to both bands; mice homozygous for the mutation give rise to only a 417 bp band; and mice homozygous for the wild-type allele have a 142 bp band. Produces only a band. Tissue samples were obtained from the tails of the animals and the PCR procedure of Example 1 was utilized for such evaluation.
[0105]
PS-1^nP264LMice that are homozygous for the allele (ie, PS-1^{nP264L / nP264L}) Are heterozygous mice (PS-1^{nP264L / +}) Is a mouse homozygous for the humanized APP gene (disclosed in PCT Publication No. W096 / 34097, published October 31, 1996; which is fully incorporated herein by reference). Produced by crossing with The resulting offspring of the generation are then PS-1^nP264LIt was determined to be heterozygous for the allele, and also heterozygous for the humanized APP gene (data not shown); progeny of these generations were then used in crosses, The resulting offspring is PS-1^nP264LWas determined (using the PCR procedure outlined above) to be homozygous for the humanized APP gene (generation offspring from this litter were PS- 1^nP264LHeterozygous for allele / homozygous for humanized APP gene; PS-1^nP264LSome were found to be heterozygous for the allele / heterozygous for the humanized APP gene-double homozygotes were not found due to the limited number of children obtained from this litter ). Subsequent mating is PS-1^{nP264L / nP264L}× APP^{NLh / NLh}Spawned mice.
[0106]
PS-1^nP264LMice homozygous for the allele are also mice heterozygous (PS-1^{nP264L / +}). In one set of crosses, 6 homozygotes were found in 27 offspring, well within the expected 25% homozygous recovery from heterozygous crossings. .
[0107]
Thus, and also based on the various breeding approaches disclosed above, the substantially normal viability and fetal survival of this animal is evident.
Example 6: Cutting out PGK-neo cassette
PBS185 plasmid DNA encoding Cre recombinase (Sauer et al., New BioL, 1990, 2, 441-449, which is incorporated herein by reference in its entirety), is pronuclear injected (pronuclear). Injection), PS-1^{nP261L / +}XCD-1 was introduced into one-cell embryos generated from the cross. Since the plasmid was circular, integration of the DNA into the genome had a very low frequency of occurrence. Transient expression of the DNA producing Cre recombinase excised the PGK-neo cassette in early embryos. The injected embryos were transferred to pseudopregnant females. Excision of the PGK-neo cassette was confirmed by determining the progeny of the progeny. These mice are PS-1^{P264L / +}Named PS-1^{P264L / P264L}Negotiated to generate mice.
[0108]
Since the neomycin selectable marker reduced transcription of the PS-1 gene, the PGK-neo gene was excised by genetic recombination at the adjacent loxP site following transient expression of Cre recombinase. PS-1^{nP264L / +}One-cell embryos (n = 154) created by crossing with xCD-1 were injected with pBS185 plasmid DNA and implanted into pseudopregnant females. Loss of the PGK-neo gene was evaluated in progeny as a 219 base pair fragment by PCR using a pair of X8F and X8R primers as described in Example 5. The mutant PS-1 allele from which the neomycin selectable marker has been excised is PS-1^P264LIt was named. Excision was successful in one founder mouse and heterozygous (PS-1^{P264L / +}) And homozygous (PS-1)^{P264L / P264L}A) mouse strain was generated. PS-1^{P264L / +}Mice were tested with Tg2576 mice and APP to further study the effect of the P264L mutation on Aβ production and deposition.^{NLh / NLh}I was crossed with a mouse.
Example 7: Northern and Western blots
PS-1 2-6 months old^{+ / +}, PS-1^{nP264L / nP264L}And PS-1^{P264L / P264L}Mice were used to evaluate PS-1 mRNA and protein levels. Total RNA was extracted from half the brain by homogenization in RNAzol B (Tel-Test, Friendswood, TX). Messenger RNA was selected on Oligotex columns (Qiagen, Valencia, CA). Equal volumes of mRNA were mixed with loading buffer (Northern MAX-Gly, Ambion, Austin, Tex.), Heated to 50 ° C. for 30 minutes, separated on a 0.7% agarose gel, and transferred to a nylon membrane. PS-1 mRNA represents the 3 'end (nucleotides 1083-1428) of human PS-1 cloned into the pGEM-T vector (Promega, Madison, WI).³²It was detected by a P-dUTP-labeled riboprobe. The same blot was hybridized with a GAPDH probe (Ambion) for normalization. To visualize the mRNA, the membrane was exposed to a phosphor screen, scanned with a Storm 840 Phosphormager, and concentration measurements were performed with ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).
[0109]
Half of the brain contains 10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS, 0.25% deoxycholate, 0.25% NP-40, and a protease inhibitor (5 mM PMSF, 10 μg / ml aprotinin, 10 μg / Ml leupeptin / 10 μg / ml pepstatin) and homogenized in 2.5 ml of buffer (Lee et al., Nature Med., 1997, 3, 756-760). Protein concentration was determined by the BCA assay (Pierce, Rockford, IL). Whole brain lysates were mixed with reducing loading buffer and heated at 37 ° C. for 45 minutes. 50 μg of total protein from each sample was separated by electrophoresis on a NuPAGE 10% polyacrylamide gel (Novex, San Diego, CA) and transferred to nitrocellulose. The membrane was blocked in Tris buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk at 4 ° C. overnight. PS-1 was detected by a rabbit polyclonal antibody diluted in the same solution. The C-terminal fragment was detected at a dilution of 1: 2000 with antibody B17.2 (DeStrooper et al., J. Biol. Chem., 1997, 272, 3590-3598). B17.2 was generated against amino acid residues 300-315 (EGDPEAQRRVSKNSKY; SEQ ID NO: 17) in the hydrophilic loop domain of human PS-1. The N-terminal fragment was detected by a 1: 500 dilution of CP160. CP160 was made using a 6 × histidine-tagged N-terminal fragment of human PS-1 (amino acids 1-80) expressed in bacteria using the pQE-9 plasmid (Qiagen). Synthetic PS-1 N-terminal fragment was purified using Ni-NTA agarose (Qiagen) and SDS-PAGE. Peptides were excised from acrylamide gels for injection into rabbits. The IgG fraction of CP160 was affinity purified and used for blotting. Primary antibodies were detected by horseradish peroxidase-labeled anti-rabbit secondary antibody (New England Biolabs, Beverly, MA). Blots were reacted with chemiluminescent reagents (LumiGLO, New England Biolabs) and exposed to Hyperfilm (Amersham, Arlington Heights, IL). Films were scanned and density measurements were performed with RFLP2.1 software (Scanalytics, Fairfax, VA).
[0110]
Northern blot analysis showed a 3.1 kb band for PS-1 mRNA. PS-1 mRNA levels were normalized with respect to GAPDH mRNA levels for differences in loading. PS-1^{nP264L / nP264L}The presence of the PGK-neo gene in mice resulted in PS-1 mRNA levels that were 20% of wild type levels (data not shown). PS-1^{P264L / P264L}MRNA levels in mice are PS-1^{+ / +}100% of mice (data not shown). Thus, removal of the PGK-neo cassette returned PS-1 mRNA levels to normal levels.
[0111]
Western blotting shows an N-terminal PS-1 fragment of about 30 kDa when using antibody CP160 and a C-terminal PS-fragment of about 20 kDa when using antibody B 17.2 in all three genotypes. One fragment was shown. Blotting with CP160 in which the antigen had been pre-absorbed eliminated the N-terminal band of approximately 30 kDa (data not shown). The specificity of B17.2 for the C-terminal PS-1 fragment has been previously shown (De Strooper et al., J. Biol. Chem., 1997, 272, 3590-3598). PS-1^{nP264L / nP264L}Due to reduced PS-1 mRNA levels in mice, PS-1 protein levels were also reduced to about 15-20% of normal levels (data not shown). PS-1^{P264L / P264L}Despite normal mRNA expression of mutant PS-1 in mice, PS-1 protein levels were reduced to about 50% (data not shown). Both N-terminal and C-terminal fragments were reduced to a similar extent. These data are PS-1^P264LMutations are shown to affect PS-1 protein levels, either through translation, processing of the full-length PS-1 protein, or through effects on the stability of the truncated fragment. Reports have described various reductions in N-terminal fragments and / or accumulation of holoproteins due to some FAD mutations in PS-1 (Mercken et al., FEBS Lett., 1996, 389, 297-303; Murayayama). See, et al., Neurosci. Lett., 1997, 229, 61-64; Levey et al., Ann. Neurol, 1997, 41, 742-753; Murayama et al., Prog. Neuro-Psychol. Takahashi et al., Neurosci. Lett., 1999, 260, 121-124). . In contrast, it was found that various PS-1 FAD mutations did not cause reduced fragmentation in FAD patients, in transfected cells, and in transgenic or gene-targeted mice (Hendriks et al., NeuroReport, 1997, 8, 1717-1721; Lee, et al., Nature Med, 1997, 3, 756-760; Podlisny et al., Neurobiol. Dis., 1997, 3, 325-3. Levesque et al., Molec. Med, 1999, 5, 542-5, et al., Nature Med, 1999, 5, 101-106. Vanderhoeven et al., Neurosci. Lett., 1999, 274, 183-186; Borchelt et al., Neuron, 1996, 17, 1005-1013; Duff et al., Nature, 13,10- Citron et al., Nature Med, 1997, 3, 67-72; Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581). The level of N-terminal fragment reduction due to the P264L mutation in transfected PC12 cells is about 35-40% of that of wild-type PS-1 (Murayama et al., Prog. Neuro-Psychopharmacol. Biol. Psychiatr., 1999, 23, 905-913), PS-1^{+ / +}PS1 in comparison with mouse^{P264L / P264L}Consistent with the degree of reduction seen in mice.
Example 8: Aβ40 specific and 42 specific ELISA
Predepositing dual gene targeting mice (1-6 months APP)^{NLh / NLh}Mouse, 5 month APP^{NLh / NLh}× PS-1^{P2 64L / +}Mouse and 1-2 month APP^{NLh / NLh}× PS-1^{P264L / P264L}Mice) and half of the brains from Tg2576 mice (Hsiao et al., Science, 1996, 274, 99-102) showing a tendency to deposit for 2-4 months are frozen on dry ice and stored at -70 ° C. Was done. PS-1^{P264L / +}Tg2576 mice showing a tendency to deposit (Tg2576 × PS-1 at 2-4 months) crossed with mice^{+ / +}Mouse, 2 months Tg2576 × PS-1^{P264L / +}Mice, and one month of Tg2576 × PS-1^{P264L / P264L}Half of the brain from mouse) was further prepared in a similar manner. Half of the brain was homogenized in 4 ml of 0.2% diethylamine and 50 mM NaCl and centrifuged at 100,000 × g. The supernatant was neutralized to pH 8 with 2M Tris-HCl, analyzed for protein concentration by the BCA method (Pierce, Rockford, IL), and 5% fetal clone serum (HyClone, Logan, UT). Was diluted 1: 1 with a 1% skim milk powder in TBS solution. Aβ42-specific ELISA was performed as previously described (Savage et al., J Neurosci., 1998, 18, 1743-1752). The Aβ40-specific ELISA was modified (Savage et al., J. Neurosci., 1998, 18, 1743-1755), the capture antibody was 6E10 (Senetek, Napa, CA) and the detection antibody was selective for Aβ40. (BioSource International, Camarillo, CA). ELISA signals were reported as nanograms of Aβ per milligram of total extracted protein, based on a standard curve generated using Aβ40 or 42 (Bachem, King of Prussia, PA).
[0112]
Table 2 shows PS-1^P264LBefore the mutation appears in Aβ deposits, APP^{NLh / NLh}Figure 4 shows the effect on Aβ40 and Aβ42 levels in mouse brain. PS-1^P264LThe mutation had no significant effect on Aβ40 levels. One copy of PS-1^P264LThe mutation slightly increased Aβ42, but the effect of the mutation was APP^{NLh / NLh}× PS-1^{+ / +}APP compared to mice^{NLh / NLh}× PS1^{P264L / P264L}It was significant only in mice. This increase in Aβ42 levels is due to APP^{NLh / NLh}× PS-1^{+ / +}And APP^{NLh / NLh}× PS-1^{P264L / +}APP compared to mice^{NLh / NLh}× PS-1^{P264L / P264L}Caused a significant increase in the ratio of Aβ42 to Aβ40 in mice. Tg2576 mouse is APP^{NLh / NLh}× PS-1^{P264L / P264L}Although they had significantly more Aβ40 and Aβ42 than mice, the Aβ42 / 40 ratio was higher for Tg2576 and APP.^{NLh / NLh}× PS-1^{+ / +}Similar in mice.
[0113]
Table 3 shows that PS-1^P264LFigure 9 shows the effect of the mutation on Aβ40 and Aβ42 levels in the brain of Tg2576 mice before the appearance of Aβ deposition. PS-1^P264LThe mutation had no significant effect on Aβ40 levels. One copy of PS-1^P264LThe mutation slightly increased Aβ42, but the effect of the mutation was Tg2576 × PS-1^{+ / +}Compared to mice, Tg2576 × PS-1^{P264L / P264L}It was significant only in mice. This increase in Aβ42 levels was due to Tg2576 × PS-1^{+ / +}Tg2576 × PS-1 compared to mouse^{P264L / P264L}In mice, it caused a significant increase in the ratio of Aβ42 to Aβ40. Thus, PS-1^P264LThe effects of mutations on Aβ levels were Tg2576 and APP^{NLh / NLh}Similar for mice.
[0114]
[Table 2]

[0115]
[Table 3]

[0116]
Example 9: Immunohistochemistry and histology
PS-1^{P264L / P264L}Mice were examined at 12 months of age. PS-1, 3, 6, 9, 12, 15 and 18 months of age^{+ / +}, PS-1^{P264L / +}Or PS-1^{P264L / P264L}APP^{NLh / NLh}The mice were evaluated. A further mouse tested was Tg2576 and PS-1^{+ / +}, PS-1^{P264L / +}Or PS-1^{P264L / P264L}At 1, 2, 4, 6, 9, 12, 15, and 18 months of age. Other Tg2576 mice maintained by crossing to C57B6 / SJL mice were also examined at 6, 9, 12, 15, 18, and 21 months of age. Mice were perfused with Ringer's solution, the brain was removed, and bisected. Half of each brain was soaked in 70% ethanol and 150 mM NaCl for 48 hours, embedded with paraffin, and sectioned at 10 μm in the sagittal plane. A set of 16 sections were taken at 200 μm intervals and stained by immunohistochemistry to show Aβ deposition. The antibodies used were 1153, a rabbit polyclonal antibody raised against amino acids 1-28 of human Aβ (Savage et al., Neuroscience, 1994, 60, 607619) and monoclonal antibodies 4G8 and 6E10 (Senetek). . Sections were pretreated with 80% formic acid for 4G8, no pretreatment for 1153 and 6E10, and reacted overnight with the primary antibody at a 1: 1,000 dilution. Antibodies were conjugated using a biotinylated secondary antibody (1: 100), ligated using horseradish peroxidase-labeled streptavidin (BioGenex, San Ramon, Calif.), And nickel-enhanced 3,3′-diamino. Visualized using benzylidene. Non-transgenic mice and pre-absorbed primary antiserum served as staining controls. Additional sets of sections were stained with Thioflavin S and examined under a fluorescence microscope.
[0117]
Plaque load in the neocortex was quantified in a set of 16 sections stained with the 1153 antibody using the CastGrid system (Olympus, Copenhagen, Demmark). The volume of the neocortex and the percent volume of the neocortex occupied by Aβ deposits were determined by point counting (Weibel et al., (1979) Stereological methods, vol. 1: practical methods for biologics. Academic Press). Representative results are shown in Table 4.
[0118]
Table 4. Aβ plaque content (% volume content) in neocortex at 6 months of age

Extracellular Aβ deposition was due to PS-1^{+ / +}PS-1^{P264L / +}Or PS-1^{P264L / P264L}Was significantly accelerated in the Tg2576 mouse. Tg2576 × PS-1^{P264L / +}In mice, Aβ deposition was not observed at 2 months of age but was present at 4 months of age. Tg2576 x PS-1^{P264L / P264L}In mice, Aβ deposition was absent at one month of age but was present at two months of age. Tg2576 × PS-1^{+ / +}Mice did not show Aβ deposition until 6 months of age, the amount corresponding to that seen in 6 month old Tg2576 mice maintained on a C57B6 / SJL background. The amount of Aβ plaque visualized with the 1153 antibody was higher for the older Tg2576 × PS-1.^{P264L / +}And Tg2576 × PS-1^{P264L / P264L}Dramatically increased in mouse neocortex (data not shown). 4 months old Tg2576 x PS-1^{P264L / +}Mice and 2 months old Tg2576 × PS-1^{P264L / P264L}Mice have multiple deposits stained with 4G8 or Thioflavin S, indicating that the earliest deposits contain compact fibrillar amyloid.
[0119]
Tg2576 × PS-1^{P264L / P264L}In addition to the accelerated deposition seen in mice, another difference was the area distribution of the deposition. Tg2576 x PS-1 at 6 months of age^{P264L / P264L}Mice, 9 months old Tg2576 × pS-1^{P261L / +}When comparing mice and 18 month old Tg2576 (C57B6 / SJL background) mice, the density of deposition was similar in telencephalic structures (data not shown). However, Tg2576 × pS-1^{P261L / PI61L}In mouse subcortical structures, the amount of Aβ deposition was Tg2576 × PS-1^{P264L / +}And in Tg2576 mice (data not shown).
[0120]
APP^{NLh / NLh}Aβ deposition in mice was assessed up to 22 months of age. No evidence of deposition was found. Similarly, PS-1 which is wild type for mouse APP^{P264L / P264L}No deposits were found in mice at 12 months of age. Extremely rare Aβ deposition was observed with APP using both 1153 antibody and Thioflavin S.^{NLh / NLh}× PS-1^{P264L / +}It was observed at 12 months of age in the cortex of mice. At 18 months of age, Aβ deposition in these mice was more numerous and larger.
[0121]
2 copies of PS-1^P264LMutation is APP^{NLh / NLh}× PS-1^{P264L / P264L}This resulted in an increase in Aβ42 in mice (Table 2), resulting in Aβ deposition at an early age. APP^{NLh / NLh}× PS-1^{P264L / P264L}In mice, Aβ deposition was not found at 3 months of age but was present at 6 months. Deposition is APP^{NLh / NLh}× PS-1^{P264L / P264L}In mice, it increased with age.
[0122]
The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference in their entirety.
[0123]
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
[Brief description of the drawings]
FIG.
1 is an illustration specifically illustrating the general principle of gene targeting according to the present invention.
FIG. 2
FIG. 4 is a set of mouse PS-1 genomic clone maps prepared using the Flash [Flash] ™ non-radioactive gene mapping kit according to the invention. The letter abbreviations for restriction endonucleases are as follows: E, EcoRI; X, XbaI; H, HindIII; B, BamHI; Xh, XhoI.
FIG. 3
1 is a representative restriction map used to illustrate the Flash [TM] restriction mapping method according to the invention.
FIG. 4
FIG. 2 is a diagram specifically illustrating a strategy for positioning exons 7 and 8 on a PS-1 restriction map according to the present invention.
FIG. 5
1 is a pair of genetic maps specifically illustrating the relationship between exon 8 of PS-1 and a pPS1-8-TV replacement vector according to the invention. The letter abbreviations for restriction endonucleases are as follows: E, EcoRI; X, XbaI; H, HindIII; B, BamHI; Xh, XhoI, N, NotI.
FIG. 6
The plasmid pPNTIox according to the invention²Is an illustration for specifically explaining the construction of.
FIG. 7
1 is an illustration specifically illustrating the construction of plasmid pPS1-XH16 according to the present invention.
FIG. 8
1 is an illustration specifically illustrating the construction of plasmid pPS1-XB1 according to the present invention.
FIG. 9
1 is an illustration specifically illustrating the construction of plasmids pPS1-X15 and pPS1-X2 according to the present invention.
FIG. 10
1 is an illustration specifically illustrating the construction of plasmid pPS1-X319 according to the present invention.
FIG. 11
FIG. 2 is a diagram specifically illustrating the restriction mapping of homologous 5 ′ arms from plasmids pPS1-X15 and pPS1-X2 according to the invention.
FIG.
FIG. 2 is a pair of restriction maps for the homologous 3 'and 5' arms of PS1 according to the invention.
FIG. 13
FIG. 2 is a partial sequence of exon 8 of PS-1 illustrating the P264L mutation and the base change to effect the addition of an AflII restriction endonuclease site according to the invention.
FIG. 14
1 is an illustration specifically illustrating the construction of plasmid pPS1-XB85 according to the present invention.
FIG.
1 is an illustration specifically illustrating the construction of plasmid pPS1-206 according to the present invention.
FIG.
1 is an illustration specifically illustrating the construction of plasmid pPS1-360 according to the present invention.
FIG.
1 is an illustration specifically illustrating the construction of plasmid pPNT3'413 according to the present invention.
FIG.
1 is an illustration specifically illustrating the construction of a plasmid pPS1-8-TV according to the present invention.
FIG.
FIG. 2 is an illustration specifically illustrating a strategy for detecting homologous recombination in mouse PS1 according to the present invention. The letter abbreviations for restriction endonucleases are as follows: E, EcoRI; X, XbaI; N, NotI; H, HindIII; B, BamHI; A, ApaI; Af, AflII; Sc, ScaI; K, KpnI; Hc, HincII.

Claims (76)

Target transgenic non-human heterozygous for human mutation of presenilin-1 (PS-1 gene), human familial Alzheimer's disease (FAD) Swedish mutation and human FAD mutation comprising humanized Aβ gene Mammals. Target transgenic non-human homozygous for human mutation of presenilin-1 (PS-1 gene), human familial Alzheimer's disease (FAD) Swedish mutation and human FAD mutation comprising humanized Aβ gene Mammals. The mammal according to claim 1, wherein the mutation of the PS-1 gene is P264L. The mammal according to claim 2, wherein the mutation of the PS-1 gene is P264L. 2. The mammal according to claim 1, wherein said mammal is a rodent. The mammal according to claim 5, wherein the mammal is a mouse. 3. The mammal according to claim 2, wherein said mammal is a rodent. The mammal according to claim 7, wherein the mammal is a mouse. 2. The reproductive offspring of a mammal according to claim 1, wherein said mutant PS-1 gene is expressed. 3. The reproductive offspring of a mammal according to claim 2, wherein said mutant PS-1 gene is expressed. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
2. Aβ in a tissue sample from a mammal according to claim 1, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
3. Aβ in a tissue sample from a mammal according to claim 2, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
10. Aβ in a tissue sample from a mammal according to claim 9, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
11. The Aβ in a tissue sample from a mammal according to claim 10, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. The method according to claim 11, wherein the tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 13. The method of claim 12, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 14. The method of claim 13, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 15. The method of claim 14, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. A method for identifying a compound for treating Alzheimer's disease, comprising:
2. The Aβ peptide in a tissue sample from a mammal according to claim 1, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. A method for identifying a compound for treating Alzheimer's disease, comprising:
3. The Aβ peptide in a tissue sample from a mammal according to claim 2, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. A method for identifying a compound for treating Alzheimer's disease, comprising:
10. The Aβ peptide in a tissue sample from a mammal according to claim 9, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. A method for identifying a compound for treating Alzheimer's disease, comprising:
The Aβ peptide in a tissue sample from a mammal according to claim 10, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. 20. The method of claim 19, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 21. The method of claim 20, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 22. The method of claim 21, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 23. The method of claim 22, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 20. A method of treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 19 to the individual. 21. A method of treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 20 to the individual. 22. A method for treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 21 to the individual. 23. A method for treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 22 to the individual. A compound identified by the method of claim 11. A compound identified by the method of claim 12. A compound identified by the method of claim 13. A compound identified by the method of claim 14. A compound identified by the method of claim 19. A compound identified by the method of claim 20. A compound identified by the method of claim 21. A compound identified by the method of claim 22. A target transgenic non-human mammal heterozygous for a human familial Alzheimer's disease (FAD) mutation comprising a human mutation of presenilin-1 (PS-1 gene) and a human transgenic for the Swedish APP695. A target transgenic non-human mammal homozygous for a human familial Alzheimer's disease (FAD) mutation comprising a human mutation of presenilin-1 (PS-1 gene) and a human transgenic for the Swedish APP695. 40. The mammal of claim 39, wherein said mutation of said PS-1 gene is P264L. 41. The mammal of claim 40, wherein said mutation of said PS-1 gene is P264L. 40. The mammal of claim 39, wherein said mammal is a rodent. 44. The mammal according to claim 43, wherein said mammal is a mouse. 41. The mammal according to claim 40, wherein said mammal is a rodent. 46. The mammal according to claim 45, wherein said mammal is a mouse. 40. The reproductive offspring of a mammal according to claim 39, wherein said mutant PS-1 gene is expressed. 41. The reproductive offspring of a mammal according to claim 40, wherein said mutant PS-1 gene is expressed. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
40. Aβ in a tissue sample from a mammal according to claim 39, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
42. Aβ in a tissue sample from a mammal according to claim 40, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
48. Aβ in a tissue sample from a mammal according to claim 47, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. A method of screening a compound for the ability to reduce in vivo levels of an Aβ peptide, comprising:
49. Aβ in a tissue sample from a mammal according to claim 48, comprising the steps of: a) administering the compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. The above method, wherein decreasing the amount of the peptide is indicative of a compound having the ability to reduce the in vivo level of the Aβ peptide. 50. The method of claim 49, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 51. The method of claim 50, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 52. The method of claim 51, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 53. The method of claim 52, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. A method for identifying a compound for treating Alzheimer's disease, comprising:
The Aβ peptide in a tissue sample from a mammal according to claim 39, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. A method for identifying a compound for treating Alzheimer's disease, comprising:
42. The Aβ peptide in a tissue sample from a mammal according to claim 40, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. A method for identifying a compound for treating Alzheimer's disease, comprising:
48. The Aβ peptide in a tissue sample from a mammal according to claim 47, comprising the steps of: a) administering a compound to the mammal; and b) determining the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. A method for identifying a compound for treating Alzheimer's disease, comprising:
49. The Aβ peptide in a tissue sample from a mammal according to claim 48, comprising the steps of: a) administering a compound to the mammal; and b) measuring the amount of Aβ peptide in the tissue sample. Said method wherein a decrease in the amount of is indicative of a compound that can be used to treat Alzheimer's disease. 58. The method of claim 57, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 59. The method of claim 58, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 60. The method of claim 59, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 61. The method of claim 60, wherein said tissue sample is selected from the group consisting of brain tissue, non-brain tissue and body fluid. 58. A method of treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 57 to said individual. 59. A method of treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 58 to said individual. 60. A method of treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 59 to the individual. 61. A method of treating an individual suspected of having Alzheimer's disease, comprising administering an effective Alzheimer's disease therapeutic amount of a compound identified by the method of claim 60 to the individual. 50. A compound identified by the method of claim 49. A compound identified by the method of claim 50. A compound identified by the method of claim 51. A compound identified by the method of claim 52. 58. A compound identified by the method of claim 57. A compound identified by the method of claim 58. 60. A compound identified by the method of claim 59. A compound identified by the method of claim 60.

Images